Silicon containing modified nucleotide analogs

ABSTRACT

Disclosed herein, inter alia, are silicon containing cleavable linkers and compounds for use in nucleic acid sequencing.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.63/305,987 filed Feb. 2, 2022, which is incorporated herein by referencein entirety and for all purposes.

BACKGROUND

DNA sequencing is a fundamental tool in biological and medical research;it is an essential technology for the paradigm of personalized precisionmedicine. Among various new DNA sequencing methods, sequencing bysynthesis (SBS) is the leading method for realizing the goal of the$1,000 genome. Accordingly, there is a need for modified nucleotides andnucleosides that are effectively recognized as substrates by DNApolymerases, that are efficiently and accurately incorporated intogrowing DNA chains during SBS. Disclosed herein, inter alia, aresolutions to these and other problems in the art.

BRIEF SUMMARY

In an aspect is provided a compound having the formula

B is a divalent nucleobase. R¹ is a 5′-nucleoside protecting group,5′-O-nucleoside protecting group, monophosphate moiety, polyphosphatemoiety, or nucleic acid moiety. R² and R³ are independently hydrogen,halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl,—CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂,—OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, polymerase-compatible cleavablemoiety, or a —O-polymerase-compatible cleavable moiety. R⁴ is adetectable moiety. L¹⁰⁰ is a divalent linker including

R⁵ and R⁶ are independently hydrogen, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. R⁷, R⁸, and R⁹ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.

In another aspect is provided a compound having the formula

B is a divalent nucleobase. R¹ is a 5′-nucleoside protecting group,5′-O-nucleoside protecting group, monophosphate moiety, polyphosphatemoiety, or nucleic acid moiety. R² is hydrogen, halogen, —CCl₃, —CBr₃,—CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br,—OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl, or apolymerase-compatible cleavable moiety. R³ is a —O-polymerase compatiblecleavable moiety having the formula:

R⁴ is a detectable moiety. L¹⁰⁰ is a divalent linker. R⁵ and R⁶ areindependently hydrogen, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. R⁷,R⁸, and R⁹ are independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. R¹⁰ is substituted or unsubstituted alkyl.

In an aspect is provided a nucleic acid polymerase complex including anucleic acid polymerase, wherein the nucleic acid polymerase is bound toa compound as described herein, including embodiments.

In an aspect is provided a method for sequencing a nucleic acid,including: (i) incorporating in series with a nucleic acid polymerase(e.g., within a reaction vessel), one of four different compounds asdescribed herein into a primer to create an extension strand, whereinthe primer is hybridized to the nucleic acid and wherein each of thefour different compounds comprises a unique detectable label; and (ii)detecting the unique detectable label of each incorporated compound, soas to thereby identify each incorporated compound in the extensionstrand, thereby sequencing the nucleic acid; wherein each of the fourdifferent compounds is independently a compound as described herein,including embodiments.

In an aspect is provided a method of sequencing nucleic acid including:(i) providing a nucleic acid template hybridized to a primer; and (ii)extending the primer hybridized to said nucleic acid template with acompound as described herein, including embodiments; and (iii)identifying the compound, so as to sequence the nucleic acid.

In another aspect is provided a method of incorporating a compound intoa primer, the method including combining a polymerase, a primerhybridized to nucleic acid template and the compound within a reactionvessel and allowing the polymerase to incorporate the compound into theprimer thereby forming an extended primer, wherein the compound is acompound as described herein, including embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Some modified nucleotides that have a linear disulfidemoiety result in one or more reactive thiols (highlighted with dashedcircle) in FIG. 1A. These reactive thiols may have downstreamcomplications. For example, a free thiol can serve as a reducing agentand prematurely remove the reversible terminator and or the linker in alabeled modified nucleotide. The thiol can also interact with functionalgroups present on the label and modulate the detectability of the label(e.g., change the fluorescence emission profile of a fluorophore).Additionally, the remainder of the linker connected to the label mayreact with the surrounding environment (e.g., the surface of theflowcell or other biomolecules) and cause an increase in backgroundsignal. FIGS. 1B-1C provide embodiments of the Si-containing cleavablelinkers described herein, where the linkers yield thioaldehydesfollowing cleavage, and optionally a terminal hydroxyl moiety.

DETAILED DESCRIPTION

The aspects and embodiments described herein relate to novel nucleotideanalogues.

I. Definitions

All patents, patent applications, articles and publications mentionedherein, both supra and infra, are hereby expressly incorporated hereinby reference in their entireties.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Various scientificdictionaries that include the terms included herein are well known andavailable to those in the art. Although any methods and materialssimilar or equivalent to those described herein find use in the practiceor testing of the disclosure, some preferred methods and materials aredescribed. Accordingly, the terms defined immediately below are morefully described by reference to the specification as a whole. It is tobe understood that this disclosure is not limited to the particularmethodology, protocols, and reagents described, as these may vary,depending upon the context in which they are used by those of skill inthe art. The following definitions are provided to facilitateunderstanding of certain terms used frequently herein and are not meantto limit the scope of the present disclosure.

As used herein, the singular terms “a”, “an”, and “the” include theplural reference unless the context clearly indicates otherwise.Reference throughout this specification to, for example, “oneembodiment”, “an embodiment”, “another embodiment”, “a particularembodiment”, “a related embodiment”, “a certain embodiment”, “anadditional embodiment”, or “a further embodiment” or combinationsthereof means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present disclosure. Thus, the appearances of theforegoing phrases in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedcarbon chain (or carbon), or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include mono-, di- andmultivalent radicals. The alkyl may include a designated number ofcarbons (e.g., C₁-C₁₀ means one to ten carbons). In embodiments, thealkyl is fully saturated. In embodiments, the alkyl is monounsaturated.In embodiments, the alkyl is polyunsaturated. Alkyl is an uncyclizedchain. Examples of saturated hydrocarbon radicals include, but are notlimited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, sec-butyl, methyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1˜and 3-propynyl,3-butynyl, and the higher homologs and isomers. An alkoxy is an alkylattached to the remainder of the molecule via an oxygen linker (—O—). Analkyl moiety may be an alkenyl moiety. An alkyl moiety may be an alkynylmoiety. An alkenyl includes one or more double bonds. An alkynylincludes one or more triple bonds.

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms, with those groupshaving 10 or fewer carbon atoms being preferred herein. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms. The term “alkenylene,” byitself or as part of another substituent, means, unless otherwisestated, a divalent radical derived from an alkene. The term “alkynylene”by itself or as part of another substituent, means, unless otherwisestated, a divalent radical derived from an alkyne. The term “alkynylene”by itself or as part of another substituent, means, unless otherwisestated, a divalent radical derived from an alkyne. In embodiments, thealkylene is fully saturated. In embodiments, the alkylene ismonounsaturated. In embodiments, the alkylene is polyunsaturated. Analkenylene includes one or more double bonds. An alkynylene includes oneor more triple bonds.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcombinations thereof, including at least one carbon atom and at leastone heteroatom (e.g., O, N, P, Si, and S), and wherein the nitrogen andsulfur atoms may optionally be oxidized, and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) (e.g., O, N, S, Si, or P)may be placed at any interior position of the heteroalkyl group or atthe position at which the alkyl group is attached to the remainder ofthe molecule. Heteroalkyl is an uncyclized chain. Examples include, butare not limited to: —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—S—CH₂, —S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,—CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and —CN. Up to two or threeheteroatoms may be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃. A heteroalkyl moiety may include one heteroatom (e.g.,O, N, S, Si, or P). A heteroalkyl moiety may include two optionallydifferent heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moietymay include three optionally different heteroatoms (e.g., O, N, S, Si,or P). A heteroalkyl moiety may include four optionally differentheteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may includefive optionally different heteroatoms (e.g., O, N, S, Si, or P). Aheteroalkyl moiety may include up to 8 optionally different heteroatoms(e.g., O, N, S, Si, or P). The term “heteroalkenyl,” by itself or incombination with another term, means, unless otherwise stated, aheteroalkyl including at least one double bond. A heteroalkenyl mayoptionally include more than one double bond and/or one or more triplebonds in additional to the one or more double bonds. The term“heteroalkynyl,” by itself or in combination with another term, means,unless otherwise stated, a heteroalkyl including at least one triplebond. A heteroalkynyl may optionally include more than one triple bondand/or one or more double bonds in additional to the one or more triplebonds. In embodiments, the heteroalkyl is fully saturated. Inembodiments, the heteroalkyl is monounsaturated. In embodiments, theheteroalkyl is polyunsaturated.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— represents both —C(O)₂R′- and —R′C(O)₂—. As describedabove, heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR′, NR′R″, —OR′, —SR′, and/or —SO₂R′. Where “heteroalkyl”is recited, followed by recitations of specific heteroalkyl groups, suchas —NR′R″ or the like, it will be understood that the terms heteroalkyland —NR′R″ are not redundant or mutually exclusive. Rather, the specificheteroalkyl groups are recited to add clarity. Thus, the term“heteroalkyl” should not be interpreted herein as excluding specificheteroalkyl groups, such as —NR′R″ or the like. The term“heteroalkenylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from a heteroalkene.The term “heteroalkynylene” by itself or as part of another substituent,means, unless otherwise stated, a divalent radical derived from aheteroalkyne. In embodiments, the heteroalkylene is fully saturated. Inembodiments, the heteroalkylene is monounsaturated. In embodiments, theheteroalkylene is polyunsaturated. A heteroalkenylene includes one ormore double bonds. A heteroalkynylene includes one or more triple bonds.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl andheterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively. In embodiments, the cycloalkyl is fully saturated. Inembodiments, the cycloalkyl is monounsaturated. In embodiments, thecycloalkyl is polyunsaturated. In embodiments, the heterocycloalkyl isfully saturated. In embodiments, the heterocycloalkyl ismonounsaturated. In embodiments, the heterocycloalkyl ispolyunsaturated.

In embodiments, the term “cycloalkyl” means a monocyclic, bicyclic, or amulticyclic cycloalkyl ring system. In embodiments, monocyclic ringsystems are cyclic hydrocarbon groups containing from 3 to 8 carbonatoms, where such groups can be saturated or unsaturated, but notaromatic. In embodiments, cycloalkyl groups are fully saturated. Inembodiments, a bicyclic or multicyclic cycloalkyl ring system refers tomultiple rings fused together or multiple spirocyclic rings wherein atleast one of the fused or spirocyclic rings is a cycloalkyl ring andwherein the multiple rings are attached to the parent molecular moietythrough any carbon atom contained within a cycloalkyl ring of themultiple rings.

In embodiments, a cycloalkyl is a cycloalkenyl. The term “cycloalkenyl”is used in accordance with its plain ordinary meaning. In embodiments, acycloalkenyl is a monocyclic, bicyclic, or a multicyclic cycloalkenylring system. In embodiments, a bicyclic or multicyclic cycloalkenyl ringsystem refers to multiple rings fused together or multiple spirocyclicrings wherein at least one of the fused or spirocyclic rings is acycloalkenyl ring and wherein the multiple rings are attached to theparent molecular moiety through any carbon atom contained within acycloalkenyl ring of the multiple rings.

In embodiments, the term “heterocycloalkyl” means a monocyclic,bicyclic, or a multicyclic heterocycloalkyl ring system. In embodiments,heterocycloalkyl groups are fully saturated. In embodiments, a bicyclicor multicyclic heterocycloalkyl ring system refers to multiple ringsfused together or multiple spirocyclic rings wherein at least one of thefused or spirocyclic rings is a heterocycloalkyl ring and wherein themultiple rings are attached to the parent molecular moiety through anyatom contained within a heterocycloalkyl ring of the multiple rings.

In embodiments, the term “cycloalkyl” means a monocyclic, bicyclic, or amulticyclic cycloalkyl ring system. In embodiments, monocyclic ringsystems are cyclic hydrocarbon groups containing from 3 to 8 carbonatoms, where such groups can be saturated or unsaturated, but notaromatic. In embodiments, cycloalkyl groups are fully saturated. Inembodiments, a bicyclic or multicyclic cycloalkyl ring system refers tomultiple rings fused together or multiple spirocyclic rings wherein atleast one of the fused or spirocyclic rings is a cycloalkyl ring andwherein the multiple rings are attached to the parent molecular moietythrough any carbon atom contained within a cycloalkyl ring of themultiple rings.

In embodiments, a cycloalkyl is a cycloalkenyl. The term “cycloalkenyl”is used in accordance with its plain ordinary meaning. In embodiments, acycloalkenyl is a monocyclic, bicyclic, or a multicyclic cycloalkenylring system. In embodiments, a bicyclic or multicyclic cycloalkenyl ringsystem refers to multiple rings fused together or multiple spirocyclicrings wherein at least one of the fused rings or spirocyclic rings is acycloalkenyl ring and wherein the multiple rings are attached to theparent molecular moiety through any carbon atom contained within acycloalkenyl ring of the multiple rings.

In embodiments, the term “heterocycloalkyl” means a monocyclic,bicyclic, or a multicyclic heterocycloalkyl ring system. In embodiments,the term “heterocycloalkyl” means a monocyclic, bicyclic, or amulticyclic heterocycloalkyl ring system. In embodiments,heterocycloalkyl groups are fully saturated. A bicyclic or multicyclicheterocycloalkyl ring system refers to multiple rings fused togetherwherein at least one of the fused rings is a heterocycloalkyl ring andwherein the multiple rings are attached to the parent molecular moietythrough any atom contained within a heterocycloalkyl ring of themultiple rings.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring and wherein the multiple rings are attached to theparent molecular moiety through any carbon atom contained within an arylring of the multiple rings. The term “heteroaryl” refers to aryl groups(or rings) that contain at least one heteroatom such as N, O, or S,wherein the nitrogen and sulfur atoms are optionally oxidized, and thenitrogen atom(s) are optionally quaternized. Thus, the term “heteroaryl”includes fused ring heteroaryl groups (i.e., multiple rings fusedtogether wherein at least one of the fused rings is a heteroaromaticring and wherein the multiple rings are attached to the parent molecularmoiety through any atom contained within a heteroaromatic ring of themultiple rings). A 5,6-fused ring heteroarylene refers to two ringsfused together, wherein one ring has 5 members and the other ring has 6members, and wherein at least one ring is a heteroaryl ring. Likewise, a6,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 6 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylenerefers to two rings fused together, wherein one ring has 6 members andthe other ring has 5 members, and wherein at least one ring is aheteroaryl ring. A heteroaryl group can be attached to the remainder ofthe molecule through a carbon or heteroatom. Non-limiting examples ofaryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl,pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl,purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl,pyrimidyl, benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran,isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl,quinoxalinyl, quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituentsfor each of the above noted aryl and heteroaryl ring systems areselected from the group of acceptable substituents described below. An“arylene” and a “heteroarylene,” alone or as part of anothersubstituent, mean a divalent radical derived from an aryl andheteroaryl, respectively. A heteroaryl group substituent may be —O—bonded to a ring heteroatom nitrogen.

Spirocyclic rings are two or more rings wherein adjacent rings areattached through a single atom. The individual rings within spirocyclicrings may be identical or different. Individual rings in spirocyclicrings may be substituted or unsubstituted and may have differentsubstituents from other individual rings within a set of spirocyclicrings. Possible substituents for individual rings within spirocyclicrings are the possible substituents for the same ring when not part ofspirocyclic rings (e.g., substituents for cycloalkyl or heterocycloalkylrings). Spirocyclic rings may be substituted or unsubstitutedcycloalkyl, substituted or unsubstituted cycloalkylene, substituted orunsubstituted heterocycloalkyl or substituted or unsubstitutedheterocycloalkylene and individual rings within a spirocyclic ring groupmay be any of the immediately previous list, including having all ringsof one type (e.g., all rings being substituted heterocycloalkylenewherein each ring may be the same or different substitutedheterocycloalkylene). When referring to a spirocyclic ring system,heterocyclic spirocyclic rings means a spirocyclic rings wherein atleast one ring is a heterocyclic ring and wherein each ring may be adifferent ring. When referring to a spirocyclic ring system, substitutedspirocyclic rings means that at least one ring is substituted and eachsubstituent may optionally be different.

The symbol “

” denotes the point of attachment of a chemical moiety to the remainderof a molecule or chemical formula.

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl,”“heterocycloalkyl,” “aryl,” and “heteroaryl”) includes both substitutedand unsubstituted forms of the indicated radical. Preferred substituentsfor each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″,—ONR′R″, —NR′C(O)NR″NR′″R″″, —CN, —NO₂, —NR′SO₂R″, —NR′C(O)R″,—NR′C(O)—OR″, —NR′OR″, in a number ranging from zero to (2m′+1), wherem′ is the total number of carbon atoms in such radical. R, R′, R″, R′″,and R″″ each preferably independently refer to hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl (e.g., aryl substituted with 1-3 halogens),substituted or unsubstituted heteroaryl, substituted or unsubstitutedalkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When acompound described herein includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″,and R″″ group when more than one of these groups is present. When R′ andR″ are attached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ includes, but is not limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″, —ONR′R″,—NR′C(O)NR″NR′″R″″, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy,and fluoro(C₁-C₄)alkyl, —NR′SO₂R″, —NR′C(O)R″, —NR′C(O)—OR″, —NR′OR″, ina number ranging from zero to the total number of open valences on thearomatic ring system; and where R′, R″, R′″, and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl. When a compound described herein includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″, and R″″ groups when more than one of these groupsis present.

As used herein, the term “associated” or “associated with” can mean thattwo or more species are identifiable as being co-located at a point intime. An association can mean that two or more species are or werewithin a similar container. An association can be an informaticsassociation, where for example digital information regarding two or morespecies is stored and can be used to determine that one or more of thespecies were co-located at a point in time. An association can also be aphysical association.

Substituents for rings (e.g., cycloalkyl, heterocycloalkyl, aryl,heteroaryl, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene) may be depicted as substituents on the ring rather thanon a specific atom of a ring (commonly referred to as a floatingsubstituent). In such a case, the substituent may be attached to any ofthe ring atoms (obeying the rules of chemical valency) and in the caseof fused rings or spirocyclic rings, a substituent depicted asassociated with one member of the fused rings or spirocyclic rings (afloating substituent on a single ring), may be a substituent on any ofthe fused rings or spirocyclic rings (a floating substituent on multiplerings). When a substituent is attached to a ring, but not a specificatom (a floating substituent), and a subscript for the substituent is aninteger greater than one, the multiple substituents may be on the sameatom, same ring, different atoms, different fused rings, differentspirocyclic rings, and each substituent may optionally be different.Where a point of attachment of a ring to the remainder of a molecule isnot limited to a single atom (a floating substituent), the attachmentpoint may be any atom of the ring and in the case of a fused ring orspirocyclic ring, any atom of any of the fused rings or spirocyclicrings while obeying the rules of chemical valency. Where a ring, fusedrings, or spirocyclic rings contain one or more ring heteroatoms and thering, fused rings, or spirocyclic rings are shown with one more floatingsubstituents (including, but not limited to, points of attachment to theremainder of the molecule), the floating substituents may be bonded tothe heteroatoms. Where the ring heteroatoms are shown bound to one ormore hydrogens (e.g., a ring nitrogen with two bonds to ring atoms and athird bond to a hydrogen) in the structure or formula with the floatingsubstituent, when the heteroatom is bonded to the floating substituent,the substituent will be understood to replace the hydrogen, whileobeying the rules of chemical valency.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   -   (A) oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,        —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH,        —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,        —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,        —OCCl₃, —OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂,        —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, unsubstituted alkyl        (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl), unsubstituted        heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered        heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted        cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or C₅-C₆        cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8        membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or        5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g.,        C₆-C₁₀ aryl, C₁₀ aryl, or phenyl), or unsubstituted heteroaryl        (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl,        or 5 to 6 membered heteroaryl), and    -   (B) alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl),        heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered        heteroalkyl, or 2 to 4 membered heteroalkyl), cycloalkyl (e.g.,        C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or C₅-C₆ cycloalkyl),        heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6        membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl),        aryl (e.g., C₆-C₁₀ aryl, C₁₀ aryl, or phenyl), heteroaryl (e.g.,        5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to        6 membered heteroaryl), substituted with at least one        substituent selected from:        -   (i) oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,            —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,            —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,            —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,            —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCl₃, —OCHCl₂,            —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F,            —N₃, unsubstituted alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or            C₁-C₄ alkyl), unsubstituted heteroalkyl (e.g., 2 to 8            membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4            membered heteroalkyl), unsubstituted cycloalkyl (e.g., C₃-C₈            cycloalkyl, C₃-C₆ cycloalkyl, or C₅-C₆ cycloalkyl),            unsubstituted heterocycloalkyl (e.g., 3 to 8 membered            heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to            6 membered heterocycloalkyl), unsubstituted aryl (e.g.,            C₆-C₁₀ aryl, C₁₀ aryl, or phenyl), or unsubstituted            heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9            membered heteroaryl, or 5 to 6 membered heteroaryl),            and (ii) alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄            alkyl), heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to            6 membered heteroalkyl, or 2 to 4 membered heteroalkyl),            cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or            C₅-C₆ cycloalkyl), heterocycloalkyl (e.g., 3 to 8 membered            heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to            6 membered heterocycloalkyl), aryl (e.g., C₆-C₁₀ aryl, C₁₀            aryl, or phenyl), heteroaryl (e.g., 5 to 10 membered            heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered            heteroaryl), substituted with at least one substituent            selected from:            -   (a) oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,                —CHBr₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —OH, —NH₂,                —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,                —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,                —NHC(O)OH, —NHOH, —OCCl₃, —OCBr₃, —OCl₃, —OCHCl₂,                —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I,                —OCH₂F, —N₃, unsubstituted alkyl (e.g., C₁-C₈ alkyl,                C₁-C₆ alkyl, or C₁-C₄ alkyl), unsubstituted heteroalkyl                (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered                heteroalkyl, or 2 to 4 membered heteroalkyl),                unsubstituted cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆                cycloalkyl, or C₅-C₆ cycloalkyl), unsubstituted                heterocycloalkyl (e.g., 3 to 8 membered                heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5                to 6 membered heterocycloalkyl), unsubstituted aryl                (e.g., C₆-C₁₀ aryl, C₁₀ aryl, or phenyl), or                unsubstituted heteroaryl (e.g., 5 to 10 membered                heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6                membered heteroaryl), and            -   (b) alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄                alkyl), heteroalkyl (e.g., 2 to 8 membered heteroalkyl,                2 to 6 membered heteroalkyl, or 2 to 4 membered                heteroalkyl), cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆                cycloalkyl, or C₅-C₆ cycloalkyl), heterocycloalkyl                (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered                heterocycloalkyl, or 5 to 6 membered heterocycloalkyl),                aryl (e.g., C₆-C₁₀ aryl, C₁₀ aryl, or phenyl),                heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9                membered heteroaryl, or 5 to 6 membered heteroaryl),                substituted with at least one substituent selected from:                oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,                —CHI₂, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —OH, —NH₂, —COOH,                —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,                —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH,                —NHOH, —OCCl₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂,                —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂T, —OCH₂F, —N₃,                unsubstituted alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or                C₁-C₄ alkyl), unsubstituted heteroalkyl (e.g., 2 to 8                membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2                to 4 membered heteroalkyl), unsubstituted cycloalkyl                (e.g., C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or C₅-C₆                cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to                8 membered heterocycloalkyl, 3 to 6 membered                heterocycloalkyl, or 5 to 6 membered heterocycloalkyl),                unsubstituted aryl (e.g., C₆-C₁₀ aryl, C₁₀ aryl, or                phenyl), or unsubstituted heteroaryl (e.g., 5 to 10                membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to                6 membered heteroaryl).

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl.

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted phenyl, and each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 6membered heteroaryl.

In some embodiments, each substituted group described in the compoundsherein is substituted with at least one substituent group. Morespecifically, in some embodiments, each substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, substituted heteroaryl, substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene described in the compounds herein are substituted with atleast one substituent group. In other embodiments, at least one or allof these groups are substituted with at least one size-limitedsubstituent group. In other embodiments, at least one or all of thesegroups are substituted with at least one lower substituent group.

In other embodiments of the compounds herein, each substituted orunsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl,each substituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and/or each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl. In someembodiments of the compounds herein, each substituted or unsubstitutedalkylene is a substituted or unsubstituted C₁-C₂₀ alkylene, eachsubstituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 20 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₈cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 8 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 10 membered heteroarylene.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted phenyl, and/or each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 6membered heteroaryl. In some embodiments, each substituted orunsubstituted alkylene is a substituted or unsubstituted C₁-C₈ alkylene,each substituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 8 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 7 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedphenylene, and/or each substituted or unsubstituted heteroarylene is asubstituted or unsubstituted 5 to 6 membered heteroarylene. In someembodiments, the compound (e.g., nucleotide analogue) is a chemicalspecies set forth in the Examples section, claims, embodiments, figures,or tables below.

In embodiments, a substituted or unsubstituted moiety (e.g., substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, and/orsubstituted or unsubstituted heteroarylene) is unsubstituted (e.g., isan unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,unsubstituted heteroaryl, unsubstituted alkylene, unsubstitutedheteroalkylene, unsubstituted cycloalkylene, unsubstitutedheterocycloalkylene, unsubstituted arylene, and/or unsubstitutedheteroarylene, respectively). In embodiments, a substituted orunsubstituted moiety (e.g., substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, and/or substituted or unsubstituted heteroarylene) issubstituted (e.g., is a substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl,substituted heteroaryl, substituted alkyl ene, substitutedheteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene, respectively).

In embodiments, a substituted moiety (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, substituted heteroaryl, substitutedalkylene, substituted heteroalkylene, substituted cycloalkylene,substituted heterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,wherein if the substituted moiety is substituted with a plurality ofsubstituent groups, each substituent group may optionally be different.In embodiments, if the substituted moiety is substituted with aplurality of substituent groups, each substituent group is different.

In embodiments, a substituted moiety (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, substituted heteroaryl, substitutedalkylene, substituted heteroalkylene, substituted cycloalkylene,substituted heterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one size-limited substituentgroup, wherein if the substituted moiety is substituted with a pluralityof size-limited substituent groups, each size-limited substituent groupmay optionally be different. In embodiments, if the substituted moietyis substituted with a plurality of size-limited substituent groups, eachsize-limited substituent group is different.

In embodiments, a substituted moiety (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, substituted heteroaryl, substitutedalkylene, substituted heteroalkylene, substituted cycloalkylene,substituted heterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited subsituent group, or lower substituent group; wherein ifthe substituted moiety is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups, each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, if the substituted moiety is substituted witha plurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups, each lower substituentgroup is different.

In embodiments, a substituted moiety (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, substituted heteroaryl, substitutedalkylene, substituted heteroalkylene, substituted cycloalkylene,substituted heterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted moiety is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, if the substituted moiety is substituted witha plurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent group isdifferent.

Certain compounds of the present disclosure possess asymmetric carbonatoms (optical or chiral centers) or double bonds; the enantiomers,racemates, diastereomers, tautomers, geometric isomers, stereoisometricforms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present disclosure. The compounds ofthe present disclosure do not include those that are known in art to betoo unstable to synthesize and/or isolate. The present disclosure ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers. As used herein, the term “isomers” refers to compounds havingthe same number and kind of atoms, and hence the same molecular weight,but differing in respect to the structural arrangement or configurationof the atoms. The term “tautomer,” as used herein, refers to one of twoor more structural isomers which exist in equilibrium and which arereadily converted from one isomeric form to another. It will be apparentto one skilled in the art that certain compounds of this disclosure mayexist in tautomeric forms, all such tautomeric forms of the compoundsbeing within the scope of the disclosure.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of thedisclosure.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this disclosure. The compounds of the presentdisclosure may also contain unnatural proportions of atomic isotopes atone or more of the atoms that constitute such compounds. For example,the compounds may be radiolabeled with radioactive isotopes, such as forexample tritium (³H), iodine-125 (¹²⁵I), or carbon-14 (¹⁴C). Allisotopic variations of the compounds of the present disclosure, whetherradioactive or not, are encompassed within the scope of the presentdisclosure.

It should be noted that throughout the application that alternatives arewritten in Markush groups, for example, each amino acid position thatcontains more than one possible amino acid. It is specificallycontemplated that each member of the Markush group should be consideredseparately, thereby comprising another embodiment, and the Markush groupis not to be read as a single unit.

“Analog,” “analogue” or “derivative” is used in accordance with itsplain ordinary meaning within Chemistry and Biology and refers to achemical compound that is structurally similar to another compound(i.e., a so-called “reference” compound) but differs in composition,e.g., in the replacement of one atom by an atom of a different element,or in the presence of a particular functional group, or the replacementof one functional group by another functional group, or the absolutestereochemistry of one or more chiral centers of the reference compound.Accordingly, an analog is a compound that is similar or comparable infunction and appearance but not in structure or origin to a referencecompound.

The terms “a” or “an,” as used in herein means one or more. In addition,the phrase “substituted with a[n],” as used herein, means the specifiedgroup may be substituted with one or more of any or all of the namedsubstituents. For example, where a group, such as an alkyl or heteroarylgroup, is “substituted with an unsubstituted C₁-C₂₀ alkyl, orunsubstituted 2 to 20 membered heteroalkyl,” the group may contain oneor more unsubstituted C₁-C₂₀ alkyls, and/or one or more unsubstituted 2to 20 membered heteroalkyls.

Moreover, where a moiety is substituted with an R substituent, the groupmay be referred to as “R-substituted.” Where a moiety is R-substituted,the moiety is substituted with at least one R substituent and each Rsubstituent is optionally different. Where a particular R group ispresent in the description of a chemical genus (such as Formula (I)), aRoman alphabetic symbol may be used to distinguish each appearance ofthat particular R group. For example, where multiple R¹³ substituentsare present, each R¹³ substituent may be distinguished as R^(13A),R^(13B), R^(13C), R^(13D), etc., wherein each of R^(13A), R^(13B),R^(13C), R^(13D), etc. is defined within the scope of the definition of10³ and optionally differently.

A “detectable agent,” “detectable compound,” “detectable label,” or“detectable moiety” is a substance (e.g., element), molecule, orcomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, chemical, magnetic resonance imaging, or other physicalmeans. For example, detectable agents include ¹⁸F, ³²P, ³³P, ⁴⁵Ti, ⁴⁷Sc,⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷As, ⁸⁶Y, ⁹⁰Y, ⁸⁹Sr, ⁸⁹Zr,⁹⁴Tc, ⁹⁴Tc, ^(99m)Tc, ⁹⁹Mo, ¹⁰⁵Pd, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ¹²³I, ¹²⁴I,¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁵⁴, ¹⁵⁸Gd, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho,¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Ac, ²¹¹At,²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra, ²²⁵Ac, Cr, V, Mn, Fe, Co, Ni, Cu, La,Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, ³²P, fluorophore(e.g., fluorescent dyes), modified oligonucleotides (e.g., moietiesdescribed in PCT/US2015/022063, which is incorporated herein byreference), electron-dense reagents, enzymes (e.g., as commonly used inan ELISA), biotin, digoxigenin, paramagnetic molecules, paramagneticnanoparticles, ultrasmall superparamagnetic iron oxide (“USPIO”)nanoparticles, USPIO nanoparticle aggregates, superparamagnetic ironoxide (“SPIO”) nanoparticles, SPIO nanoparticle aggregates,monochrystalline iron oxide nanoparticles, monochrystalline iron oxide,nanoparticle contrast agents, liposomes or other delivery vehiclescontaining Gadolinium chelate (“Gd-chelate”) molecules, Gadolinium,radioisotopes, radionuclides (e.g., carbon-11, nitrogen-13, oxygen-15,fluorine-18, rubidium-82), fluorodeoxyglucose (e.g., fluorine-18labeled), any gamma ray emitting radionuclides, positron-emittingradionuclide, radiolabeled glucose, radiolabeled water, radiolabeledammonia, biocolloids, microbubbles (e.g., including microbubble shellsincluding albumin, galactose, lipid, and/or polymers; microbubble gascore including air, heavy gas(es), perfluorcarbon, nitrogen,octafluoropropane, perflexane lipid microsphere, perflutren, etc.),iodinated contrast agents (e.g., iohexol, iodixanol, ioversol,iopamidol, ioxilan, iopromide, diatrizoate, metrizoate, ioxaglate),barium sulfate, thorium dioxide, gold, gold nanoparticles, goldnanoparticle aggregates, fluorophores, two-photon fluorophores, orhaptens and proteins or other entities which can be made detectable,e.g., by incorporating a radiolabel into a peptide or antibodyspecifically reactive with a target peptide. In embodiments, adetectable moiety is a moiety (e.g., monovalent form) of a detectableagent.

The terms “fluorophore” or “fluorescent agent” or “fluorescent dye” areused interchangeably and refer to a substance, compound, agent (e.g., adetectable agent), or composition (e.g., compound) that can absorb lightat one or more wavelengths and re-emit light at one or more longerwavelengths, relative to the one or more wavelengths of absorbed light.Examples of fluorophores that may be included in the compounds andcompositions described herein include fluorescent proteins, xanthenederivatives (e.g., fluorescein, rhodamine, Oregon green, eosin, or Texasred), cyanine and derivatives (e.g., cyanine, indocarbocyanine,oxacarbocyanine, thiacarbocyanine, or merocyanine), napththalenederivatives (e.g., dansyl or prodan derivatives), coumarin andderivatives, oxadiazole derivatives (e.g., pyridyloxazole,nitrobenzoxadiazole or benzoxadiazole), anthracene derivatives (e.g.,anthraquinones, DRAQ5, DRAQ7, or CyTRAK Orange), pyrene derivatives(e.g., cascade blue and derivatives), oxazine derivatives (e.g., Nilered, Nile blue, cresyl violet, or oxazine 170), acridine derivatives(e.g., proflavin, acridine orange, acridine yellow), arylmethinederivatives (e.g., auramine, crystal violet, or malachite green),tetrapyrrole derivatives (e.g., porphin, phthalocyanine, bilirubin), CFDye™ DRAQ™ CyTRAK™, BODIPY™, Alexa Fluor™, DyLight Fluor™, Atto™,Tracy™, FluoProbes™, Abberior Dyes™ DY™ dyes, MegaStokes Dyes™, SulfoCy™, Seta™ dyes, SeTau™ dyes, Square Dyes™, Quasar™ dyes, Cal Fluor™dyes, SureLight Dyes™, PerCP™ Phycobilisomes™ APC™ APCXL™ RPE™ and/orBPE™. A fluorescent moiety is a radical of a fluorescent agent. Theemission from the fluorophores can be detected by any number of methods,including but not limited to, fluorescence spectroscopy, fluorescencemicroscopy, fluorimeters, fluorescent plate readers, infrared scanneranalysis, laser scanning confocal microscopy, automated confocalnanoscanning, laser spectrophotometers, fluorescent-activated cellsorters (FACS), image-based analyzers and fluorescent scanners (e.g.,gel/membrane scanners). In embodiments, the fluorophore is an aromatic(e.g., polyaromatic) moiety having a conjugated π-electron system. Inembodiments, the fluorophore is a fluorescent dye moiety, that is, amonovalent fluorophore.

Radioactive substances (e.g., radioisotopes) that may be used as imagingand/or labeling agents in accordance with the embodiments of thedisclosure include, but are not limited to, ¹⁸F, ³²P, ³³P, ⁴⁵Ti, ⁴⁷Sc,⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷As, ⁸⁶Y, ⁹⁰Y, ⁸⁹Sr, ⁸⁹Zr,⁹⁴Tc, ⁹⁴Tc, ^(99m)Tc, ⁹⁹Mo, ¹⁰⁵Pd, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ¹²³I, ¹²⁴I,¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁵⁴⁻¹⁵⁸Gd, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho,¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Ay, ²¹¹At,²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra and ²²⁵Ac. Paramagnetic ions that maybe used as additional imaging agents in accordance with the embodimentsof the disclosure include, but are not limited to, ions of transitionand lanthanide metals (e.g., metals having atomic numbers of 21-29, 42,43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni,Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.

Examples of detectable agents include imaging agents, includingfluorescent and luminescent substances, molecules, or compositions,including, but not limited to, a variety of organic or inorganic smallmolecules commonly referred to as “dyes,” “labels,” or “indicators.”Examples include fluorescein, rhodamine, acridine dyes, Alexa dyes, andcyanine dyes. In embodiments, the detectable moiety is a fluorescentmolecule (e.g., acridine dye, cyanine dye, fluorine dye, oxazine dye,phenanthridine dye, or rhodamine dye). In embodiments, the detectablemoiety is a fluorescent molecule (e.g., acridine dye, cyanine dye,fluorine dye, oxazine dye, phenanthridine dye, or rhodamine dye). Inembodiments, the detectable moiety is a fluorescent moiety orfluorescent dye moiety.

In embodiments, the detectable moiety is a fluorescein isothiocyanatemoiety, tetramethylrhodamine-5-(and 6)-isothiocyanate moiety, Cy2moiety, Cy3 moiety, Cy5 moiety, Cy7 moiety,4′,6-diamidino-2-phenylindole moiety, Hoechst 33258 moiety, Hoechst33342 moiety, Hoechst 34580 moiety, propidium-iodide moiety, or acridineorange moiety. In embodiments, the detectable moiety is a Indo-1, Casaturated moiety, Indo-1 Ca2+ moiety, Cascade Blue BSA pH 7.0 moiety,Cascade Blue moiety, LysoTracker Blue moiety, Alexa 405 moiety,LysoSensor Blue pH 5.0 moiety, LysoSensor Blue moiety, DyLight 405moiety, DyLight 350 moiety, BFP (Blue Fluorescent Protein) moiety, Alexa350 moiety, 7-Amino-4-methylcoumarin pH 7.0 moiety, Amino Coumarinmoiety, AMCA conjugate moiety, Coumarin moiety,7-Hydroxy-4-methylcoumarin moiety, 7-Hydroxy-4-methylcoumarin pH 9.0moiety, 6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0 moiety, Hoechst33342 moiety, Pacific Blue moiety, Hoechst 33258 moiety, Hoechst33258-DNA moiety, Pacific Blue antibody conjugate pH 8.0 moiety,PO-PRO-1 moiety, PO-PRO-1-DNA moiety, POPO-1 moiety, POPO-1-DNA moiety,DAPI-DNA moiety, DAPI moiety, Marina Blue moiety, SYTOX Blue-DNA moiety,CFP (Cyan Fluorescent Protein) moiety, eCFP (Enhanced Cyan FluorescentProtein) moiety, 1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS) moiety,Indo-1, Ca free moiety, 1,8-ANS (1-Anilinonaphthalene-8-sulfonic acid)moiety, BO-PRO-1-DNA moiety, BOPRO-1 moiety, BOBO-1-DNA moiety, SYTO45-DNA moiety, evoglow-Ppl moiety, evoglow-Bs1 moiety, evoglow-Bs2moiety, Auramine 0 moiety, DiO moiety, LysoSensor Green pH 5.0 moiety,Cy 2 moiety, LysoSensor Green moiety, Fura-2, high Ca moiety, Fura-2Ca2+sup> moiety, SYTO 13-DNA moiety, YO-PRO-1-DNA moiety, YOYO-1-DNAmoiety, eGFP (Enhanced Green Fluorescent Protein) moiety, LysoTrackerGreen moiety, GFP (S65T) moiety, BODIPY FL, MeOH moiety, Sapphiremoiety, BODIPY FL conjugate moiety, MitoTracker Green moiety,MitoTracker Green FM, MeOH moiety, Fluorescein 0.1 M NaOH moiety,Calcein pH 9.0 moiety, Fluorescein pH 9.0 moiety, Calcein moiety,Fura-2, no Ca moiety, Fluo-4 moiety, FDA moiety, DTAF moiety,Fluorescein moiety, CFDA moiety, FITC moiety, Alexa Fluor 488hydrazide-water moiety, DyLight 488 moiety, 5-FAM pH 9.0 moiety, Alexa488 moiety, Rhodamine 110 moiety, Rhodamine 110 pH 7.0 moiety, AcridineOrange moiety, BCECF pH 5.5 moiety, PicoGreendsDNA quantitation reagentmoiety, SYBR Green I moiety, Rhodaminen Green pH 7.0 moiety, CyQUANTGR-DNA moiety, NeuroTrace 500/525, green fluorescent Nissl stain-RNAmoiety, DansylCadaverine moiety, Fluoro-Emerald moiety, Nissl moiety,Fluorescein dextran pH 8.0 moiety, Rhodamine Green moiety,5-(and-6)-Carboxy-2′, 7′-dichlorofluorescein pH 9.0 moiety,DansylCadaverine, MeOH moiety, eYFP (Enhanced Yellow FluorescentProtein) moiety, Oregon Green 488 moiety, Fluo-3 moiety, BCECF pH 9.0moiety, SBFI-Na+ moiety, Fluo-3 Ca2+ moiety, Rhodamine 123 MeOH moiety,FlAsH moiety, Calcium Green-1 Ca2+ moiety, Magnesium Green moiety,DM-NERF pH 4.0 moiety, Calcium Green moiety, Citrine moiety, LysoSensorYellow pH 9.0 moiety, TO-PRO-1-DNA moiety, Magnesium Green Mg2+ moiety,Sodium Green Na+ moiety, TOTO-1-DNA moiety, Oregon Green 514 moiety,Oregon Green 514 antibody conjugate pH 8.0 moiety, NBD-X moiety, DM-NERFpH 7.0 moiety, NBD-X, MeOH moiety, CI-NERF pH 6.0 moiety, Alexa 430moiety, CI-NERF pH 2.5 moiety, Lucifer Yellow, CH moiety, LysoSensorYellow pH 3.0 moiety, 6-TET, SE pH 9.0 moiety, Eosin antibody conjugatepH 8.0 moiety, Eosin moiety, 6-Carboxyrhodamine 6G pH 7.0 moiety,6-Carboxyrhodamine 6G, hydrochloride moiety, Bodipy R6G SE moiety,BODIPY R6G MeOH moiety, 6 JOE moiety, Cascade Yellow moiety, mBananamoiety, Alexa 532 moiety, Erythrosin-5-isothiocyanate pH 9.0 moiety,6-HEX, SE pH 9.0 moiety, mOrange moiety, mHoneydew moiety, Cy 3 moiety,Rhodamine B moiety, DiI moiety, 5-TAMRA-MeOH moiety, Alexa 555 moiety,DyLight 549 moiety, BODIPY TMR-X, SE moiety, BODIPY TMR-X MeOH moiety,PO-PRO-3-DNA moiety, PO-PRO-3 moiety, Rhodamine moiety, POPO-3 moiety,Alexa 546 moiety, Calcium Orange Ca2+ moiety, TRITC moiety, CalciumOrange moiety, Rhodaminephalloidin pH 7.0 moiety, MitoTracker Orangemoiety, MitoTracker Orange MeOH moiety, Phycoerythrin moiety, MagnesiumOrange moiety, R-Phycoerythrin pH 7.5 moiety, 5-TAMRA pH 7.0 moiety,5-TAMRA moiety, Rhod-2 moiety, FM 1-43 moiety, Rhod-2 Ca2+ moiety, FM1-43 lipid moiety, LOLO-1-DNA moiety, dTomato moiety, DsRed moiety,Dapoxyl (2-aminoethyl) sulfonamide moiety, Tetramethylrhodamine dextranpH 7.0 moiety, Fluor-Ruby moiety, Resorufin moiety, Resorufin pH 9.0moiety, mTangerine moiety, LysoTracker Red moiety, Lissaminerhodaminemoiety, Cy 3.5 moiety, Rhodamine Red-X antibody conjugate pH 8.0 moiety,Sulforhodamine 101 EtOH moiety, JC-1 pH 8.2 moiety, JC-1 moiety,mStrawberry moiety, MitoTracker Red moiety, MitoTracker Red, MeOHmoiety, X-Rhod-1 Ca2+ moiety, Alexa 568 moiety, 5-ROX pH 7.0 moiety,5-ROX (5-Carboxy-X-rhodamine, triethylammonium salt) moiety,BO-PRO-3-DNA moiety, BOPRO-3 moiety, BOBO-3-DNA moiety, Ethidium Bromidemoiety, ReAsH moiety, Calcium Crimson moiety, Calcium Crimson Ca2+moiety, mRFP moiety, mCherry moiety, HcRed moiety, DyLight 594 moiety,Ethidium homodimer-1-DNA moiety, Ethidiumhomodimer moiety, PropidiumIodide moiety, SYPRO Ruby moiety, Propidium Iodide-DNA moiety, Alexa 594moiety, BODIPY TR-X, SE moiety, BODIPY TR-X, MeOH moiety, BODIPY TR-Xphallacidin pH 7.0 moiety, Alexa Fluor 610 R-phycoerythrin streptavidinpH 7.2 moiety, YO-PRO-3-DNA moiety, Di-8 ANEPPS moiety,Di-8-ANEPPS-lipid moiety, YOYO-3-DNA moiety, Nile Red-lipid moiety, NileRed moiety, DyLight 633 moiety, mPlum moiety, TO-PRO-3-DNA moiety, DDAOpH 9.0 moiety, Fura Red high Ca moiety, Allophycocyanin pH 7.5 moiety,APC (allophycocyanin) moiety, Nile Blue, EtOH moiety, TOTO-3-DNA moiety,Cy 5 moiety, BODIPY 650/665-X, MeOH moiety, Alexa Fluor 647R-phycoerythrin streptavidin pH 7.2 moiety, DyLight 649 moiety, Alexa647 moiety, Fura Red Ca2+ moiety, Atto 647 moiety, Fura Red, low Camoiety, Carboxynaphthofluorescein pH 10.0 moiety, Alexa 660 moiety, Cy5.5 moiety, Alexa 680 moiety, DyLight 680 moiety, Alexa 700 moiety, FM4-64, 2% CHAPS moiety, or FM 4-64 moiety. In embodiments, the dectablemoiety is a moiety of 1,1-Diethyl-4,4-carbocyanine iodide, 1,2-Diphenylacetylene, 1,4-Diphenylbutadiene, 1,4-Diphenylbutadiyne,1,6-Diphenylhexatriene, 1,6-Diphenylhexatriene,1-anilinonaphthalene-8-sulfonic acid, 2,7-Dichlorofluorescein,2,5-DIPHENYLOXAZOLE, 2-Di-1-ASP, 2-dodecylresorufin,2-Methylbenzoxazole, 3,3-Diethylthiadicarbocyanine iodide, 4-Dimethylamino-4-Nitrostilbene, 5(6)-Carboxyfluorescein,5(6)-Carboxynaphtofluorescein, 5(6)-Carboxytetramethylrhodamine B,5-(and-6)-carboxy-2′,7′-dichlorofluorescein,5-(and-6)-carboxy-2,7-dichlorofluorescein, 5-(N-hexadecanoyl)aminoeosin,5-(N-hexadecanoyl)aminoeosin, 5-chloromethylfluorescein, 5-FAM, 5-ROX,5-TAMRA, 5-TAMRA, 6,8-difluoro-7-hydroxy-4-methylcoumarin, 6,8-difluorohydroxy-4-methylcoumarin, 6-carboxyrhodamine 6G, 6-HEX, 6-JOE, 6-JOE,6-TET, 7-aminoactinomycin D,7-Benzylamino-4-Nitrobenz-2-Oxa-1,3-Diazole, 7-Methoxycoumarin AceticAcid, 8-Benzyloxy-5,7-diphenylquinoline,8-Benzyloxy-5,7-diphenylquinoline, 9,10-Bis(Phenylethynyl)Anthracene,9,10-Diphenylanthracene, 9-METHYLCARBAZOLE, (CS)2Ir(μ-Cl)2Ir(CS)2, AAA,Acridine Orange, Acridine Orange, Acridine Yellow, Acridine Yellow,Adams Apple Red 680, Adirondack Green 520, Alexa Fluor 350, Alexa Fluor405, Alexa Fluor 430, Alexa Fluor 430, Alexa Fluor 480, Alexa Fluor 488,Alexa Fluor 488, Alexa Fluor 488 hydrazide, Alexa Fluor 500, Alexa Fluor514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 546, Alexa Fluor 555,Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 594,Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 610-R-PE, Alexa Fluor 633,Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 647, Alexa Fluor 647-R-PE,Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 680-APC, Alexa Fluor680-R-PE, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790,Allophycocyanin, AmCyanl, Aminomethylcoumarin, Amplex Gold (product),Amplex Red Reagent, Amplex UltraRed, Anthracene, APC, APC-Seta-750,AsRed2, ATTO 390, ATTO 425, ATTO 430LS, ATTO 465, ATTO 488, ATTO 490LS,ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO 550, ATTO 565, ATTO 590,ATTO 594, ATTO 610, ATTO 620, ATTO 633, ATTO 635, ATTO 647, ATTO 647N,ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740, ATTO Oxa12,ATTO Rho3B, ATTO Rho6G, ATTO Rho11, ATTO Rho12, ATTO Rho13, ATTO Rho14,ATTO Rho101, ATTO Thio12, Auramine O, Azami Green, Azami Greenmonomeric, B-phycoerythrin, BCECF, BCECF, Bexl, Biphenyl, Birch Yellow580, Blue-green algae, BO-PRO-1, BO-PRO-3, BOBO-1, BOBO-3, BODIPY 630650-X, BODIPY 650/665-X, BODIPY FL, BODIPY FL, BODIPY R6G, BODIPY TMR-X,BODIPY TR-X, BODIPY TR-X Ph 7.0, BODIPY TR-X phallacidin, BODIPY-DiMe,BODIPY-Phenyl, BODIPY-TMSCC, C3-Indocyanine, C3-Indocyanine,C3-Oxacyanine, C3-Thiacyanine Dye (EtOH), C3-Thiacyanine Dye (PrOH),C5-Indocyanine, C5-Oxacyanine, C5-Thiacyanine, C7-Indocyanine,C7-Oxacyanine, C545T, C-Phycocyanin, Calcein, Calcein red-orange,Calcium Crimson, Calcium Green-1, Calcium Orange, Calcofluor white 2MR,Carboxy SNARF-1 pH 6.0, Carboxy SNARF-1 pH 9.0,Carboxynaphthofluorescein, Cascade Blue, Cascade Yellow, Catskill Green540, CBQCA, CellMask Orange, CellTrace BODIPY TR methyl ester, CellTracecalcein violet, CellTrace™ Far Red, CellTracker Blue, CellTracker RedCMTPX, CellTracker Violet BMQC, CF405M, CF405S, CF488A, CF543, CF555,CFP, CFSE, CF™ 350, CF™ 485, Chlorophyll A, Chlorophyll B, Chromeo 488,Chromeo 494, Chromeo 505, Chromeo 546, Chromeo 642, Citrine, Citrine,ClOH butoxy aza-BODIPY, ClOH C12 aza-BODIPY, CM-H2DCFDA, Coumarin 1,Coumarin 6, Coumarin 6, Coumarin 30, Coumarin 314, Coumarin 334,Coumarin 343, Coumarine 545T, Cresyl Violet Perchlorate, CryptoLightCF₁, CryptoLight CF₂, CryptoLight CF₃, CryptoLight CF₄, CryptoLight CF5,CryptoLight CF₆, Crystal Violet, Cumarin153, Cy2, Cy3, Cy3, Cy3.5, Cy3B,Cy3B, Cy3Cy5 ET, Cy5, Cy5, Cy5.5, Cy7, Cyanine3 NHS ester, Cyanine5carboxylic acid, Cyanine5 NHS ester, Cyclotella meneghiniana Kützing,CypHer5, CypHer5 pH 9.15, CyQUANT GR, CyTrak Orange, Dabcyl SE, DAF-FM,DAMC (Weiss), dansyl cadaverine, Dansyl Glycine (Dioxane), DAPI, DAPI,DAPI, DAPI, DAPI (DMSO), DAPI (H2O), Dapoxyl (2-aminoethyl)sulfonamide,DCI, DCM, DCM, DCM (acetonitrile), DCM (MeOH), DDAO, Deep Purple,di-8-ANEPPS, DiA, Dichlorotris(1,10-phenanthroline) ruthenium(II),DiClOH C12 aza-BODIPY, DiClOHbutoxy aza-BODIPY, DiD, DiI, DiIC18(3),DiO, DiR, Diversa Cyan-FP, Diversa Green-FP, DM-NERF pH 4.0, DOCI,Doxorubicin, DPP pH-Probe 590-7.5, DPP pH-Probe 590-9.0, DPP pH-Probe590-11.0, DPP pH-Probe 590-11.0, Dragon Green, DRAQ5, DsRed, DsRed,DsRed, DsRed-Express, DsRed-Express2, DsRed-Express T1, dTomato,DY-350XL, DY-480, DY-480XL MegaStokes, DY-485, DY-485XL MegaStokes,DY-490, DY-490XL MegaStokes, DY-500, DY-500XL MegaStokes, DY-520,DY-520XL MegaStokes, DY-547, DY-549P1, DY-549P1, DY-554, DY-555, DY-557,DY-557, DY-590, DY-590, DY-615, DY-630, DY-631, DY-633, DY-635, DY-636,DY-647, DY-649P1, DY-649P1, DY-650, DY-651, DY-656, DY-673, DY-675,DY-676, DY-680, DY-681, DY-700, DY-701, DY-730, DY-731, DY-750, DY-751,DY-776, DY-782, Dye-28, Dye-33, Dye-45, Dye-304, Dye-1041, DyLight 488,DyLight 549, DyLight 594, DyLight 633, DyLight 649, DyLight 680,E2-Crimson, E2-Orange, E2-Red/Green, EBFP, ECF, ECFP, ECL Plus, eGFP,ELF 97, Emerald, Envy Green, Eosin, Eosin Y, epicocconone, EqFP611,Erythrosin-5-isothiocyanate, Ethidium bromide, ethidium homodimer-1,Ethyl Eosin, Ethyl Eosin, Ethyl Nile Blue A,Ethyl-p-Dimethylaminobenzoate, Ethyl-p-Dimethylaminobenzoate, Eu2O3nanoparticles, Eu (Soini), Eu(tta)₃DEADIT, EvaGreen, EVOblue-30, EYFP,FAD, FITC, FITC, FlAsH (Adams), Flash Red EX, FlAsH-CCPGCC,FlAsH-CCXXCC, Fluo-3, Fluo-4, Fluo-5F, Fluorescein, Fluorescein 0.1NaOH, Fluorescein-Dibase, fluoro-emerald, Fluorol 5G, FluoSpheres blue,FluoSpheres crimson, FluoSpheres dark red, FluoSpheres orange,FluoSpheres red, FluoSpheres yellow-green, FM4-64 in CTC, FM4-64 in SDS,FM 1-43, FM 4-64, Fort Orange 600, Fura Red, Fura Red Ca free, fura-2,Fura-2 Ca free, Gadodiamide, Gd-Dtpa-Bma, Gadodiamide, Gd-Dtpa-Bma,GelGreen™, GelRed™, H9-40, HcRedl, Hemo Red 720, HiLyte Fluor 488,HiLyte Fluor 555, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750,HiLyte Plus 555, HiLyte Plus 647, HiLyte Plus 750, HmGFP, Hoechst 33258,Hoechst 33342, Hoechst-33258, Hoechst-33258, Hops Yellow 560, HPTS,HPTS, HPTS, HPTS, HPTS, indo-1, Indo-1 Ca free, Ir(Cn)₂(acac),Ir(Cs)₂(acac), IR-775 chloride, IR-806, Ir-OEP—CO-Cl, IRDye® 650 Alkyne,IRDye® 650 Azide, IRDye® 650 Carboxylate, IRDye® 650 DBCO, IRDye® 650Maleimide, IRDye® 650 NHS Ester, IRDye® 680LT Carboxylate, IRDye® 680LTMaleimide, IRDye® 680LT NHS Ester, IRDye® 680RD Alkyne, IRDye® 680RDAzide, IRDye® 680RD Carboxylate, IRDye® 680RD DBCO, IRDye® 680RDMaleimide, IRDye® 680RD NHS Ester, IRDye® 700 phosphoramidite, IRDye®700DX, IRDye® 700DX, IRDye® 700DX Carboxylate, IRDye® 700DX NHS Ester,IRDye® 750 Carboxylate, IRDye® 750 Maleimide, IRDye® 750 NHS Ester,IRDye® 800 phosphoramidite, IRDye® 800CW, IRDye® 800CW Alkyne, IRDye®800CW Azide, IRDye® 800CW Carboxylate, IRDye® 800CW DBCO, IRDye® 800CWMaleimide, IRDye® 800CW NHS Ester, IRDye® 800RS, IRDye® 800RSCarboxylate, IRDye® 800RS NHS Ester, IRDye® QC-1 Carboxylate, IRDye®QC-1 NHS Ester, Isochrysis galbana—Parke, JC-1, JC-1, JOJO-1, JonamacRed Evitag T2, Kaede Green, Kaede Red, kusabira orange, Lake Placid 490,LDS 751, Lissamine Rhodamine (Weiss), LOLO-1, lucifer yellow CH, LuciferYellow CH, lucifer yellow CH, Lucifer Yellow CH Dilitium salt, LumioGreen, Lumio Red, Lumogen F Orange, Lumogen Red F300, Lumogen Red F300,LysoSensor Blue DND-192, LysoSensor Green DND-153, LysoSensor GreenDND-153, LysoSensor Yellow/Blue DND-160 pH 3, LysoSensor YellowBlueDND-160, LysoTracker Blue DND-22, LysoTracker Blue DND-22, LysoTrackerGreen DND-26, LysoTracker Red DND-99, LysoTracker Yellow HCK-123, MacounRed Evitag T2, Macrolex Fluorescence Red G, Macrolex Fluorescence Yellow10GN, Macrolex Fluorescence Yellow 10GN, Magnesium Green, MagnesiumOctaethylporphyrin, Magnesium Orange, Magnesium Phthalocyanine,Magnesium Phthalocyanine, Magnesium Tetramesitylporphyrin, MagnesiumTetraphenylporphyrin, malachite green isothiocyanate, Maple Red-Orange620, Marina Blue, mBanana, mBBr, mCherry, Merocyanine 540, Methyl green,Methyl green, Methyl green, Methylene Blue, Methylene Blue, mHoneyDew,MitoTracker Deep Red 633, MitoTracker Green FM, MitoTracker OrangeCMTMRos, MitoTracker Red CMXRos, monobromobimane, Monochlorobimane,Monoraphidium, mOrange, mOrange2, mPlum, mRaspberry, mRFP, mRFP1,mRFP1.2 (Wang), mStrawberry (Shaner), mTangerine (Shaner),N,N-Bis(2,4,6-trimethylphenyl)-3,4:9,10-perylenebis(dicarboximide),NADH, Naphthalene, Naphthalene, Naphthofluorescein, Naphthofluorescein,NBD-X, NeuroTrace 500525, Nilblau perchlorate, nile blue, Nile Blue,Nile Blue (EtOH), nile red, Nile Red, Nile Red, Nile red, Nileblue A,NIR1, NIR2, NIR3, NIR4, NIR820, Octaethylporphyrin, OH butoxyaza-BODIPY, OHC12 aza-BODIPY, Orange Fluorescent Protein, Oregon Green488, Oregon Green 488 DHPE, Oregon Green 514, Oxazin1, Oxazin 750,Oxazine 1, Oxazine 170, P4-3, P-Quaterphenyl, P-Terphenyl, PA-GFP(post-activation), PA-GFP (pre-activation), Pacific Orange,Palladium(II) meso-tetraphenyl-tetrabenzoporphyrin, PdOEPK, PdTFPP,PerCP-Cy5.5, Perylene, Perylene, Perylene bisimide pH-Probe 550-5.0,Perylene bisimide pH-Probe 550-5.5, Perylene bisimide pH-Probe 550-6.5,Perylene Green pH-Probe 720-5.5, Perylene Green Tag pH-Probe 720-6.0,Perylene Orange pH-Probe 550-2.0, Perylene Orange Tag 550, Perylene RedpH-Probe 600-5.5, Perylenediimid, Perylne Green pH-Probe 740-5.5,Phenol, Phenylalanine, pHrodo, succinimidyl ester, Phthalocyanine,PicoGreen dsDNA quantitation reagent, Pinacyanol-Iodide, Piroxicam,Platinum(II) tetraphenyltetrabenzoporphyrin, Plum Purple, PO-PRO-1,PO-PRO-3, POPO-1, POPO-3, POPOP, Porphin, PPO, Proflavin,PromoFluor-350, PromoFluor-405, PromoFluor-415, PromoFluor-488,PromoFluor-488 Premium, PromoFluor-488LSS, PromoFluor-500LSS,PromoFluor-505, PromoFluor-510LSS, PromoFluor-514LS S,PromoFluor-520LSS, PromoFluor-532, PromoFluor-546, PromoFluor-555,PromoFluor-590, PromoFluor-610, PromoFluor-633, PromoFluor-647,PromoFluor-670, PromoFluor-680, PromoFluor-700, PromoFluor-750,PromoFluor-770, PromoFluor-780, PromoFluor-840, propidium iodide,Protoporphyrin IX, PTIR475/UF, PTIR545/UF, PtOEP, PtOEPK, PtTFPP,Pyrene, QD525, QD565, QD585, QD605, QD655, QD705, QD800, QD903, QD PbS950, QDot 525, QDot 545, QDot 565, Qdot 585, Qdot 605, Qdot 625, Qdot655, Qdot 705, Qdot 800, QpyMe2, QSY 7, QSY 7, QSY 9, QSY 21, QSY 35,quinine, Quinine Sulfate, Quinine sulfate, R-phycoerythrin,R-phycoerythrin, ReAsH-CCPGCC, ReAsH-CCXXCC, Red Beads (Weiss), RedmondRed, Resorufin, resorufin, rhod-2, Rhodamin 700 perchlorate, rhodamine,Rhodamine 6G, Rhodamine 6G, Rhodamine 101, rhodamine 110, Rhodamine 123,rhodamine 123, Rhodamine B, Rhodamine B, Rhodamine Green, RhodaminepH-Probe 585-7.0, Rhodamine pH-Probe 585-7.5, Rhodamine phalloidin,Rhodamine Red-X, Rhodamine Red-X, Rhodamine Tag pH-Probe 585-7.0, RhodolGreen, Riboflavin, Rose Bengal, Sapphire, SBFI, SBFI Zero Na,Scenedesmus sp., SensiLight PBXL-1, SensiLight PBXL-3, Seta 633-NHS,Seta-633-NHS, SeTau-380-NHS, SeTau-647-NHS, Snake-Eye Red 900, SNIR1,SNIR2, SNIR3, SNIR4, Sodium Green, Solophenyl flavine 7GFE 500, SpectrumAqua, Spectrum Blue, Spectrum FRed, Spectrum Gold, Spectrum Green,Spectrum Orange, Spectrum Red, Squarylium dye III, Stains All, Stilbenderivate, Stilbene, Styry18 perchlorate, Sulfo-Cyanine3 carboxylic acid,Sulfo-Cyanine3 carboxylic acid, Sulfo-Cyanine3 NHS ester, Sulfo-Cyanine5carboxylic acid, Sulforhodamine 101, sulforhodamine 101, SulforhodamineB, Sulforhodamine G, Suncoast Yellow, SuperGlo BFP, SuperGlo GFP, SurfGreen EX, SYBR Gold nucleic acid gel stain, SYBR Green I, SYPRO Ruby,SYTO 9, SYTO 11, SYTO 13, SYTO 16, SYTO 17, SYTO 45, SYTO 59, SYTO 60,SYTO 61, SYTO 62, SYTO 82, SYTO RNASelect, SYTO RNASelect, SYTOX Blue,SYTOX Green, SYTOX Orange, SYTOX Red, T-Sapphire, Tb (Soini), tCO,tdTomato, Terrylen, Terrylendiimid, testdye, Tetra-t-Butylazaporphine,Tetra-t-Butylnaphthalocyanine, Tetracen,Tetrakis(o-Aminophenyl)Porphyrin, Tetramesitylporphyrin,Tetramethylrhodamine, tetramethylrhodamine, Tetraphenylporphyrin,Tetraphenylporphyrin, Texas Red, Texas Red DHPE, Texas Red-X,ThiolTracker Violet, Thionin acetate, TMRE, TO-PRO-1, TO-PRO-3, Toluene,Topaz (Tsien1998), TOTO-1, TOTO-3, Tris(2,2-Bipyridyl)Ruthenium(II)chloride, Tris(4,4-diphenyl-2,2-bipyridine) ruthenium(II) chloride,Tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) TMS, TRITC (Weiss),TRITC Dextran (Weiss), Tryptophan, Tyrosine, Vexl, Vybrant DyeCycleGreen stain, Vybrant DyeCycle Orange stain, Vybrant DyeCycle Violetstain, WEGFP (post-activation), WellRED D2, WellRED D3, WellRED D4,WtGFP, WtGFP (Tsien1998), X-rhod-1, Yakima Yellow, YFP, YO-PRO-1,YO-PRO-3, YOYO-1, YoYo-1, YoYo-1 dsDNA, YoYo-1 ssDNA, YOYO-3, ZincOctaethylporphyrin, Zinc Phthalocyanine, Zinc Tetramesitylporphyrin,Zinc Tetraphenylporphyrin, ZsGreen1, or ZsYellow1. In embodiments, R⁴ isa monovalent moiety of a compound described within this paragraph.

In embodiments, the detectable moiety is a moiety of a derivative of oneof the detectable moieties described immediately above, wherein thederivative differs from one of the detectable moieties immediately aboveby a modification resulting from the conjugation of the detectablemoiety to a compound described herein.

In embodiments, the detectable label is a fluorescent dye. Inembodiments, the detectable label is a fluorescent dye capable ofexchanging energy with another fluorescent dye (e.g., fluorescenceresonance energy transfer (FRET) chromophores).

The term “cyanine” or “cyanine moiety” as described herein refers to adetectable moiety containing two nitrogen groups separated by apolymethine chain. In embodiments, the cyanine moiety has 3 methinestructures (i.e., cyanine 3 or Cy3). In embodiments, the cyanine moietyhas 5 methine structures (i.e., cyanine 5 or Cy5). In embodiments, thecyanine moiety has 7 methine structures (i.e., cyanine 7 or Cy7).

Descriptions of compounds (e.g., nucleotide analogues) of the presentdisclosure are limited by principles of chemical bonding known to thoseskilled in the art. Accordingly, where a group may be substituted by oneor more of a number of substituents, such substitutions are selected soas to comply with principles of chemical bonding and to give compoundswhich are not inherently unstable and/or would be known to one ofordinary skill in the art as likely to be unstable under ambientconditions, such as aqueous, neutral, and several known physiologicalconditions. For example, a heterocycloalkyl or heteroaryl is attached tothe remainder of the molecule via a ring heteroatom in compliance withprinciples of chemical bonding known to those skilled in the art therebyavoiding inherently unstable compounds.

As used herein, the term “salt” refers to acid or base salts of thecompounds described herein. Thus, the compounds of the present inventionmay exist as salts. Non-limiting examples of such salts includehydrochlorides, hydrobromides, phosphates, sulfates, methanesulfonates,nitrates, maleates, acetates, citrates, fumarates, proprionates,tartrates (e.g., (+)-tartrates, (−)-tartrates, or mixtures thereofincluding racemic mixtures), succinates, benzoates, and salts with aminoacids such as glutamic acid, and quaternary ammonium salts (e.g., methyliodide, ethyl iodide, and the like). These salts may be prepared bymethods known to those skilled in the art. Illustrative examples ofacceptable salts are mineral acid (hydrochloric acid, hydrobromic acid,phosphoric acid, and the like) salts, organic acid (acetic acid,propionic acid, glutamic acid, citric acid and the like) salts,quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.In embodiments, compounds may be presented with a positive charge, andit is understood an appropriate counter-ion (e.g., chloride ion,fluoride ion, or acetate ion) may also be present, though not explicitlyshown. Likewise, for compounds having a negative charge (e.g.,

it is understood an appropriate counter-ion (e.g., a proton, sodium ion,potassium ion, or ammonium ion) may also be present, though notexplicitly shown. The protonation state of the compound (e.g., acompound described herein) depends on the local environment (i.e., thepH of the environment), therefore, in embodiments, the compound may bedescribed as having a moiety in a protonated state (e.g.,

or an ionic state (e.g.,

and it is understood these are interchangeable. In embodiments, thecounter-ion is represented by the symbol M (e.g., M⁺ or M⁻). The neutralforms of the compounds are preferably regenerated by contacting the saltwith a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound may differ from thevarious salt forms in certain physical properties, such as solubility inpolar solvents.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues,wherein the polymer may optionally be conjugated to a moiety that doesnot consist of amino acids. The terms apply to amino acid polymers inwhich one or more amino acid residue is an artificial chemical mimeticof a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymer.

A polypeptide, or a cell is “recombinant” when it is artificial orengineered, or derived from or contains an artificial or engineeredprotein or nucleic acid (e.g., non-natural or not wild type). Forexample, a polynucleotide that is inserted into a vector or any otherheterologous location, e.g., in a genome of a recombinant organism, suchthat it is not associated with nucleotide sequences that normally flankthe polynucleotide as it is found in nature is a recombinantpolynucleotide. A protein expressed in vitro or in vivo from arecombinant polynucleotide is an example of a recombinant polypeptide.Likewise, a polynucleotide sequence that does not appear in nature, forexample a variant of a naturally occurring gene, is recombinant.

“Hybridize” shall mean the annealing of one single-stranded nucleic acid(such as a primer) to another nucleic acid based on the well-understoodprinciple of sequence complementarity. In an embodiment, the othernucleic acid is a single-stranded nucleic acid. The propensity forhybridization between nucleic acids depends on the temperature and ionicstrength of their milieu, the length of the nucleic acids and the degreeof complementarity. The effect of these parameters on hybridization isdescribed in, for example, Sambrook J., Fritsch E. F., Maniatis T.,Molecular cloning: a laboratory manual, Cold Spring Harbor LaboratoryPress, New York (1989). As used herein, hybridization of a primer, or ofa DNA extension product, respectively, is extendable by creation of aphosphodiester bond with an available nucleotide or nucleotide analoguecapable of forming a phosphodiester bond, therewith. Those skilled inthe art understand how to estimate and adjust the stringency ofhybridization conditions such that sequences having at least a desiredlevel of complementarity will stably hybridize, while those having lowercomplementarity will not. As used herein, the term “stringent condition”refers to condition(s) under which a polynucleotide probe or primer willhybridize preferentially to its target sequence, and to a lesser extentto, or not at all to, other sequences. In some embodiments, nucleicacids, or portions thereof, that are configured to specificallyhybridize are often about 80% or more, 81% or more, 82% or more, 83% ormore, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more,89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% ormore, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more or100% complementary to each other over a contiguous portion of nucleicacid sequence. A specific hybridization discriminates over non-specifichybridization interactions (e.g., two nucleic acids that a notconfigured to specifically hybridize, e.g., two nucleic acids that are80% or less, 70% or less, 60% or less or 50% or less complementary) byabout 2-fold or more, often about 10-fold or more, and sometimes about100-fold or more, 1000-fold or more, 10,000-fold or more, 100,000-foldor more, or 1,000,000-fold or more. Two nucleic acid strands that arehybridized to each other can form a duplex, which comprises adouble-stranded portion of nucleic acid.

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments,about means within a standard deviation using measurements generallyacceptable in the art. In embodiments, about means a range extending to+/−10% of the specified value. In embodiments, about includes thespecified value.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.,chemical compounds including biomolecules or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated, however, that the resulting reaction product can beproduced directly from a reaction between the added reagents or from anintermediate from one or more of the added reagents that can be producedin the reaction mixture. The term “contacting” may include allowing twospecies to react, interact, or physically touch, wherein the two speciesmay be a compound as described herein and a protein or enzyme. In someembodiments, contacting includes allowing a compound described herein tointeract with a protein or enzyme that is involved in a signalingpathway.

The term “expression” includes any step involved in the production ofthe polypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion. Expression can be detected usingconventional techniques for detecting protein (e.g., ELISA, Westernblotting, flow cytometry, immunofluorescence, immunohistochemistry,etc.).

An “effective amount” is an amount sufficient for a compound toaccomplish a stated purpose relative to the absence of the compound(e.g., achieve the effect for which it is administered). An “activitydecreasing amount,” as used herein, refers to an amount of antagonistrequired to decrease the activity of an enzyme relative to the absenceof the antagonist. A “function disrupting amount,” as used herein,refers to the amount of antagonist required to disrupt the function ofan enzyme or protein relative to the absence of the antagonist.

“Control” or “control experiment” is used in accordance with its plainordinary meaning and refers to an experiment in which the subjects orreagents of the experiment are treated as in a parallel experimentexcept for omission of a procedure, reagent, or variable of theexperiment. In some instances, the control is used as a standard ofcomparison in evaluating experimental effects. In some embodiments, acontrol is the measurement of the activity of a protein in the absenceof a compound as described herein (including embodiments and examples).

The term “modulate” is used in accordance with its plain ordinarymeaning and refers to the act of changing or varying one or moreproperties. “Modulation” refers to the process of changing or varyingone or more properties. For example, as applied to the effects of amodulator on a target protein, to modulate means to change by increasingor decreasing a property or function of the target molecule or theamount of the target molecule.

“Nucleic acid” refers to nucleotides (e.g., deoxyribonucleotides orribonucleotides) and polymers thereof in either single-, double- ormultiple-stranded form, or complements thereof; or nucleosides (e.g.,deoxyribonucleosides or ribonucleosides). In embodiments, “nucleic acid”does not include nucleosides. The terms “polynucleotide,”“oligonucleotide,” “oligo” or the like refer, in the usual and customarysense, to a linear sequence of nucleotides. Oligonucleotides aretypically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or morenucleotides in length, up to about 100 nucleotides in length. Nucleicacids and polynucleotides are polymers of any length, including longerlengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc.In certain embodiments, the nucleic acids herein contain phosphodiesterbonds. In other embodiments, nucleic acid analogs are included that mayhave alternate backbones, comprising, e.g., phosphoramidate,phosphorothioate, phosphorodithioate, or O-methylphosphoroamiditelinkages (see, Eckstein, Oligonucleotides and Analogues: A PracticalApproach, Oxford University Press); and peptide nucleic acid backbonesand linkages. Other analog nucleic acids include those with positivebackbones; non-ionic backbones, and non-ribose backbones, includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters6 and 7, ASC Symposium Series 580, Carbohydrate Modifications inAntisense Research, Sanghui & Cook, eds. Nucleic acids containing one ormore carbocyclic sugars are also included within one definition ofnucleic acids. Modifications of the ribose-phosphate backbone may bedone for a variety of reasons, e.g., to increase the stability andhalf-life of such molecules in physiological environments or as probeson a biochip. Mixtures of naturally occurring nucleic acids and analogscan be made; alternatively, mixtures of different nucleic acid analogs,and mixtures of naturally occurring nucleic acids and analogs may bemade. A residue of a nucleic acid, as referred to herein, is a monomerof the nucleic acid (e.g., a nucleotide). The term “nucleoside” refers,in the usual and customary sense, to a glycosylamine including anucleobase and a five-carbon sugar (ribose or deoxyribose). Non-limitingexamples of nucleosides include cytidine, uridine, adenosine, guanosine,thymidine and inosine. Nucleosides may be modified at the base and/orthe sugar. The term “nucleotide” refers, in the usual and customarysense, to a single unit of a polynucleotide, i.e., a monomer.Nucleotides can be ribonucleotides, deoxyribonucleotides, or modifiedversions thereof. Examples of polynucleotides contemplated hereininclude single and double stranded DNA, single and double stranded RNA,and hybrid molecules having mixtures of single and double stranded DNAand RNA. Examples of nucleic acid, e.g., polynucleotides contemplatedherein include any types of RNA, e.g., mRNA, siRNA, miRNA, and guide RNAand any types of DNA, genomic DNA, plasmid DNA, and minicircle DNA, andany fragments thereof. The term “duplex” in the context ofpolynucleotides refers, in the usual and customary sense, to doublestrandedness. Nucleic acids can be linear or branched. For example,nucleic acids can be a linear chain of nucleotides or the nucleic acidscan be branched, e.g., such that the nucleic acids comprise one or morearms or branches of nucleotides. Optionally, the branched nucleic acidsare repetitively branched to form higher ordered structures such asdendrimers and the like. A “nucleic acid moiety” as used herein is amonovalent form of a nucleic acid. In embodiments, the nucleic acidmoiety is attached to the 3′ or 5′ position of a nucleotide ornucleoside.

Nucleic acids, including e.g., nucleic acids with a phosphorothioatebackbone, can include one or more reactive moieties. As used herein, theterm reactive moiety includes any group capable of reacting with anothermolecule, e.g., a nucleic acid or polypeptide through covalent,non-covalent or other interactions. By way of example, the nucleic acidcan include an amino acid reactive moiety that reacts with an amino acidon a protein or polypeptide through a covalent, non-covalent or otherinteraction.

As used herein, the term “template polynucleotide” refers to anypolynucleotide molecule that may be bound by a polymerase and utilizedas a template for nucleic acid synthesis. A template polynucleotide maybe a target polynucleotide. In general, the term “target polynucleotide”refers to a nucleic acid molecule or polynucleotide in a startingpopulation of nucleic acid molecules having a target sequence whosepresence, amount, and/or nucleotide sequence, or changes in one or moreof these, are desired to be determined. In general, the term “targetsequence” refers to a nucleic acid sequence on a single strand ofnucleic acid. The target sequence may be a portion of a gene, aregulatory sequence, genomic DNA, cDNA, RNA including mRNA, miRNA, rRNA,or others. The target sequence may be a target sequence from a sample ora secondary target such as a product of an amplification reaction. Atarget polynucleotide is not necessarily any single molecule orsequence. For example, a target polynucleotide may be any one of aplurality of target polynucleotides in a reaction, or allpolynucleotides in a given reaction, depending on the reactionconditions. For example, in a nucleic acid amplification reaction withrandom primers, all polynucleotides in a reaction may be amplified. As afurther example, a collection of targets may be simultaneously assayedusing polynucleotide primers directed to a plurality of targets in asingle reaction. As yet another example, all or a subset ofpolynucleotides in a sample may be modified by the addition of aprimer-binding sequence (such as by the ligation of adapters containingthe primer binding sequence), rendering each modified polynucleotide atarget polynucleotide in a reaction with the corresponding primerpolynucleotide(s). In the context of selective sequencing, “targetpolynucleotide(s)” refers to the subset of polynucleotide(s) to besequenced from within a starting population of polynucleotides.

“Nucleotide,” as used herein, refers to a nucleoside-5′-phosphate (e.g.,polyphosphate) compound, or a structural analog thereof, which can beincorporated (e.g., partially incorporated as anucleoside-5′-monophosphate or derivative thereof) by a nucleic acidpolymerase to extend a growing nucleic acid chain (such as a primer).Nucleotides may comprise bases such as adenine (A), cytosine (C),guanine (G), thymine (T), uracil (U), or analogues thereof, and maycomprise 1, 2, 3, 4, 5, 6, 7, 8, or more phosphates in the phosphategroup. Nucleotides may be modified at one or more of the base, sugar, orphosphate group. A nucleotide may have a label or tag attached (a“labeled nucleotide” or “tagged nucleotide”). In an embodiment, thenucleotide is a deoxyribonucleotide. In another embodiment, thenucleotide is a ribonucleotide. In embodiments, nucleotides comprise 3phosphate groups (e.g., a triphosphate group).

The terms also encompass nucleic acids containing known nucleotideanalogs or modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, which have similarbinding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphodiester derivativesincluding, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate(also known as phosphorothioate having double bonded sulfur replacingoxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids,phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid,methyl phosphonate, boron phosphonate, or O-methylphosphoroamiditelinkages (see, Eckstein, Oligonucleotides and Analogues: A PracticalApproach, Oxford University Press) as well as modifications to thenucleotide bases such as in 5-methyl cytidine or pseudouridine; andpeptide nucleic acid backbones and linkages. Other analog nucleic acidsinclude those with positive backbones; non-ionic backbones, modifiedsugars, and non-ribose backbones (e.g., phosphorodiamidate morpholinooligos or locked nucleic acids (LNA) as known in the art), includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters6 and 7, ASC Symposium Series 580, Carbohydrate Modifications inAntisense Research, Sanghui & Cook, eds. Nucleic acids containing one ormore carbocyclic sugars are also included within one definition ofnucleic acids. Modifications of the ribose-phosphate backbone may bedone for a variety of reasons, e.g., to increase the stability andhalf-life of such molecules in physiological environments or as probeson a biochip. Mixtures of naturally occurring nucleic acids and analogscan be made; alternatively, mixtures of different nucleic acid analogs,and mixtures of naturally occurring nucleic acids and analogs may bemade. In embodiments, the internucleotide linkages in DNA arephosphodiester, phosphodiester derivatives, or a combination of both.

In embodiments, “nucleotide analogue,” “nucleotide analog,” or“nucleotide derivative” shall mean an analogue of adenine (A), cytosine(C), guanine (G), thymine (T), or uracil (U) (that is, an analogue orderivative of a nucleotide comprising the base A, G, C, T or U),comprising a phosphate group, which may be recognized by DNA or RNApolymerase (whichever is applicable) and may be incorporated into astrand of DNA or RNA (whichever is appropriate). Examples of nucleotideanalogues include, without limitation, 7-deaza-adenine, 7-deaza-guanine,the analogues of deoxynucleotides shown herein, analogues in which alabel is attached through a cleavable linker to the 5-position ofcytosine or thymine or to the 7-position of deaza-adenine ordeaza-guanine, and analogues in which a small chemical moiety is used tocap the —OH group at the 3′-position of deoxyribose. Nucleotideanalogues and DNA polymerase-based DNA sequencing are also described inU.S. Pat. No. 6,664,079, which is incorporated herein by reference inits entirety for all purposes.

A “nucleoside” is structurally similar to a nucleotide, but is missingthe phosphate moieties. An example of a nucleoside analogue would be onein which the label is linked to the base and there is no phosphate groupattached to the sugar molecule. “Nucleoside,” as used herein, refers toa glycosyl compound consisting of a nucleobase and a 5-membered ringsugar (e.g., either ribose or deoxyribose). Nucleosides may comprisebases such as adenine (A), cytosine (C), guanine (G), thymine (T),uracil (U), or analogues thereof. Nucleosides may be modified at thebase and/or and the sugar. In an embodiment, the nucleoside is adeoxyribonucleoside. In another embodiment, the nucleoside is aribonucleoside.

The terms “bioconjugate group,” “bioconjugate reactive moiety,” and“bioconjugate reactive group” refer to a chemical moiety whichparticipates in a reaction to form a bioconjugate linker (e.g., covalentlinker). Non-limiting examples of bioconjugate groups include —NH₂,—COOH, —COOCH₃, —N-hydroxysuccinimide, -maleimide,

In embodiments, the bioconjugate reactive group may be protected (e.g.,with a protecting group). Additional examples of bioconjugate reactivegroups and the resulting bioconjugate reactive linkers may be found inthe Bioconjugate Table below:

Bioconjugate Bioconjugate reactive group 1 reactive group 2 (e.g.,electrophilic (e.g., nucleophilic Resulting bioconjugate bioconjugateBioconjugate reactive moiety) reactive moiety) reactive linker activatedesters amines/anilines carboxamides acrylamides thiols thioethers acylazides amines/anilines carboxamides acyl halides amines/anilinescarboxamides acyl halides alcohols/phenols esters acyl nitrilesalcohols/phenols esters acyl nitriles amines/anilines carboxamidesaldehydes amines/anilines imines aldehydes or ketones hydrazineshydrazones aldehydes or ketones hydroxylamines oximes alkyl halidesamines/anilines alkyl amines alkyl halides carboxylic acids esters alkylhalides thiols thioethers alkyl halides alcohols/phenols ethers alkylsulfonates thiols thioethers alkyl sulfonates carboxylic acids estersalkyl sulfonates alcohols/phenols ethers anhydrides alcohols/phenolsesters anhydrides amines/anilines carboxamides aryl halides thiolsthiophenols aryl halides amines aryl amines aziridines thiols thioethersboronates glycols boronate esters carbodiimides carboxylic acidsN-acylureas or anhydrides diazoalkanes carboxylic acids esters epoxidesthiols thioethers haloacetamides thiols thioethers haloplatinate aminoplatinum complex haloplatinate heterocycle platinum complexhaloplatinate thiol platinum complex halotriazines amines/anilinesaminotriazines halotriazines alcohols/phenols triazinyl ethershalotriazines thiols triazinyl thioethers imido esters amines/anilinesamidines isocyanates amines/anilines ureas isocyanates alcohols/phenolsurethanes isothiocyanates amines/anilines thioureas maleimides thiolsthioethers phosphoramidites alcohols phosphite esters silyl halidesalcohols silyl ethers sulfonate esters amines/anilines alkyl aminessulfonate esters thiols thioethers sulfonate esters carboxylic acidsesters sulfonate esters alcohols ethers sulfonyl halides amines/anilinessulfonamides sulfonyl halides phenols/alcohols sulfonate esters

As used herein, the term “bioconjugate” or “bioconjugate linker” refersto the resulting association between atoms or molecules of bioconjugatereactive groups. The association can be direct or indirect. For example,a conjugate between a first bioconjugate reactive group (e.g., —NH₂,—COOH, —N-hydroxysuccinimide, or -maleimide) and a second bioconjugatereactive group (e.g., sulfhydryl, sulfur-containing amino acid, amine,amine sidechain containing amino acid, or carboxylate) provided hereincan be direct, e.g., by covalent bond or linker (e.g., a first linker ofsecond linker), or indirect, e.g., by non-covalent bond (e.g.,electrostatic interactions (e.g., ionic bond, hydrogen bond, halogenbond), van der Waals interactions (e.g., dipole-dipole, dipole-induceddipole, London dispersion), ring stacking (pi effects), hydrophobicinteractions and the like). In embodiments, bioconjugates orbioconjugate linkers are formed using bioconjugate chemistry (i.e., theassociation of two bioconjugate reactive groups) including, but are notlimited to nucleophilic substitutions (e.g., reactions of amines andalcohols with acyl halides, active esters), electrophilic substitutions(e.g., enamine reactions) and additions to carbon-carbon andcarbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alderaddition). These and other useful reactions are discussed in, forexample, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons,New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, SanDiego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances inChemistry Series, Vol. 198, American Chemical Society, Washington, D.C.,1982. In embodiments, the first bioconjugate reactive group (e.g.,maleimide moiety) is covalently attached to the second bioconjugatereactive group (e.g., a sulfhydryl). In embodiments, the firstbioconjugate reactive group (e.g., haloacetyl moiety) is covalentlyattached to the second bioconjugate reactive group (e.g., a sulfhydryl).In embodiments, the first bioconjugate reactive group (e.g., pyridylmoiety) is covalently attached to the second bioconjugate reactive group(e.g., a sulfhydryl). In embodiments, the first bioconjugate reactivegroup (e.g., —N-hydroxysuccinimide moiety) is covalently attached to thesecond bioconjugate reactive group (e.g., an amine). In embodiments, thefirst bioconjugate reactive group (e.g., maleimide moiety) is covalentlyattached to the second bioconjugate reactive group (e.g., a sulfhydryl).In embodiments, the first bioconjugate reactive group (e.g.,-sulfo-N-hydroxysuccinimide moiety) is covalently attached to the secondbioconjugate reactive group (e.g., an amine). In embodiments, the firstbioconjugate reactive group (e.g., maleimide moiety) is covalentlyattached to the second bioconjugate reactive group (e.g., a sulfhydryl).In embodiments, the first bioconjugate reactive group (e.g.,-sulfo-N-hydroxysuccinimide moiety) is covalently attached to the secondbioconjugate reactive group (e.g., an amine).

The bioconjugate reactive groups can be chosen such that they do notparticipate in, or interfere with, the chemical stability of theconjugate described herein. Alternatively, a reactive functional groupcan be protected from participating in the crosslinking reaction by thepresence of a protecting group. In embodiments, the bioconjugatecomprises a molecular entity derived from the reaction of an unsaturatedbond, such as a maleimide, and a sulfhydryl group.

Useful bioconjugate reactive groups used for bioconjugate chemistriesherein include, for example: (a) carboxyl groups and various derivativesthereof including, but not limited to, N-hydroxysuccinimide esters,N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters,p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters; (b)hydroxyl groups which can be converted to esters, ethers, aldehydes,etc.; (c) haloalkyl groups wherein the halide can be later displacedwith a nucleophilic group such as, for example, an amine, a carboxylateanion, thiol anion, carbanion, or an alkoxide ion, thereby resulting inthe covalent attachment of a new group at the site of the halogen atom;(d) dienophile groups which are capable of participating in Diels-Alderreactions such as, for example, maleimido or maleimide groups; (e)aldehyde or ketone groups such that subsequent derivatization ispossible via formation of carbonyl derivatives such as, for example,imines, hydrazones, semicarbazones or oximes, or via such mechanisms asGrignard addition or alkyllithium addition; (f) sulfonyl halide groupsfor subsequent reaction with amines, for example, to form sulfonamides;(g) thiol groups, which can be converted to disulfides, reacted withacyl halides, or bonded to metals such as gold, or react withmaleimides; (h) amine or sulfhydryl groups (e.g., present in cysteine),which can be, for example, acylated, alkylated or oxidized; (i) alkenes,which can undergo, for example, cycloadditions, acylation, Michaeladdition, etc.; (j) epoxides, which can react with, for example, aminesand hydroxyl compounds; (k) phosphoramidites and other standardfunctional groups useful in nucleic acid synthesis; (1) metal siliconoxide bonding; (m) metal bonding to reactive phosphorus groups (e.g.,phosphines) to form, for example, phosphate diester bonds; (n) azidescoupled to alkynes using copper catalyzed cycloaddition click chemistry;(o) biotin conjugate can react with avidin or streptavidin to form aavidin-biotin complex or streptavidin-biotin complex.

The term “monophosphate” is used in accordance with its ordinary meaningin the arts and refers to a moiety having the formula:

or ionized forms thereof. The term “polyphosphate” refers to at leasttwo phosphate groups, having the formula:

or ionized forms thereof, wherein np is an integer of 1 or greater. Inembodiments, np is an integer from 1 to 5. In embodiments, np is aninteger from 1 to 2. In embodiments, np is 2. The term “diphosphate” isused in accordance with its ordinary meaning in the arts and refers to amoiety having the formula:

or ionized forms thereof. The term “triphosphate” is used in accordancewith its ordinary meaning in the arts and refers to a moiety having theformula:

or ionized forms thereof. In embodiments, a polyphosphate is adiphosphate. In embodiments, a polyphosphate is a triphosphate.

The term “nucleobase” or “base” as used herein refers to a purine orpyrimidine compound, or a derivative thereof, that may be a constituentof nucleic acid (i.e., DNA or RNA, or a derivative thereof). Nucleobasesare nitrogen-containing biological compounds (e.g., nitrogenous bases)found within deoxyribonucleic acid (DNA), ribonucleic acid (RNA),nucleotides, and nucleosides. The primary nucleobases are cytosine,guanine, adenine, thymine, and uracil. Adenine and guanine belong to thedouble-ringed class of molecules called purines. Cytosine, thymine, anduracil are all pyrimidines. Modified nucleobases include hypoxanthine,xanthine, 7-methylguanine, 5,6-dihyfrouracil, 5-methylcytosine, and5-hydroxymethylcytosine. A nucleobase derivative may include any of theaforementioned nucleobases including one or more substituents (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group). In embodiments, the nucleobase is a divalentpurine or pyrimidine, or derivative thereof. In embodiments, thenucleobase is a monovalent purine or pyrimidine, or derivative thereof.In embodiments, the base is a derivative of a naturally occurring DNA orRNA base (e.g., a base analogue). In embodiments, the base is ahybridizing base. In embodiments, the base hybridizes to a complementarybase. In embodiments, the base is capable of forming at least onehydrogen bond with a complementary base (e.g., adenine hydrogen bondswith thymine, adenine hydrogen bonds with uracil, guanine pairs withcytosine). Non-limiting examples of a base includes cytosine or aderivative thereof (e.g., cytosine analogue), guanine or a derivativethereof (e.g., guanine analogue), adenine or a derivative thereof (e.g.,adenine analogue), thymine or a derivative thereof (e.g., thymineanalogue), uracil or a derivative thereof (e.g., uracil analogue),hypoxanthine or a derivative thereof (e.g., hypoxanthine analogue),xanthine or a derivative thereof (e.g., xanthine analogue),7-methylguanine or a derivative thereof (e.g., 7-methylguanineanalogue), deaza-adenine or a derivative thereof (e.g., deaza-adenineanalogue), deaza-guanine or a derivative thereof (e.g., deaza-guanine),deaza-hypoxanthine or a derivative thereof, 5,6-dihydrouracil or aderivative thereof (e.g., 5,6-dihydrouracil analogue), 5-methylcytosineor a derivative thereof (e.g., 5-methylcytosine analogue), or5-hydroxymethylcytosine or a derivative thereof (e.g.,5-hydroxymethylcytosine analogue) moieties. In embodiments, the base isadenine, guanine, uracil, cytosine, thymine, hypoxanthine, xanthine,theobromine, caffeine, uric acid, or isoguanine, which may be optionallysubstituted or modified. In embodiments, the base is adenine, guanine,hypoxanthine, xanthine, theobromine, caffeine, uric acid, or isoguanine,which may be optionally substituted or modified. In embodiments, thenucleobase is

which may be optionally substituted or modified. In embodiments, thenucleobase includes

which may be optionally substituted or modified. In embodiments, thenucleobase is

In embodiments, the nucleobase is

which may be optionally substituted or modified. In embodiments, thenucleobase includes a substituted or unsubstituted propargyl aminemoiety, which may further include S—S linker, fluorophores or protectinggroup. In embodiments, the nucleobase is

which may be further substituted or modified. In embodiments, thepropargyl amine moiety may further include at least one or moreprotecting groups. In embodiments, the propargyl amine moiety mayfurther include at least one or more fluorophores. In embodiments, thepropargyl amine moiety may further be linked to a —S—S-linker, which maybe connected to at least one or more flurorophores. In embodiments, thepropargyl amine moiety may further be linked to a —S—S-linker, which maybe connected a Si-containing cleavable linkers and at least one or moreflurorophores. Purine nucleobase derivatives mimic the structure ofpurines. Examples of purine nucleobase derivatives include azathioprine,mercaptopurine, thioguanine, flubarabine, pentostatin, and cladribine.Pyrimidine nucleobase derivatives mimic the structure of metabolicpurines. Examples include, but are not limited to, 5-fluorouracil,floxuridine, cytosine arabinoside, and 6-azauracil. Nucleotidesincluding nucleobase derivatives are molecules that act like the nativenucleotides in RNA or DNA synthesis.

As used herein, the term “complementary” or “substantiallycomplementary” refers to the hybridization, base pairing, or theformation of a duplex between nucleotides or nucleic acids. For example,complementarity exists between the two strands of a double-stranded DNAmolecule or between an oligonucleotide primer and a primer binding siteon a single-stranded nucleic acid when a nucleotide (e.g., RNA or DNA)or a sequence of nucleotides is capable of base pairing with arespective cognate nucleotide or cognate sequence of nucleotides. Asdescribed herein and commonly known in the art the complementary(matching) nucleotide of adenosine (A) is thymidine (T) and thecomplementary (matching) nucleotide of guanosine (G) is cytosine (C).Thus, a complement may include a sequence of nucleotides that base pairwith corresponding complementary nucleotides of a second nucleic acidsequence. The nucleotides of a complement may partially or completelymatch the nucleotides of the second nucleic acid sequence. Where thenucleotides of the complement completely match each nucleotide of thesecond nucleic acid sequence, the complement forms base pairs with eachnucleotide of the second nucleic acid sequence. Where the nucleotides ofthe complement partially match the nucleotides of the second nucleicacid sequence only some of the nucleotides of the complement form basepairs with nucleotides of the second nucleic acid sequence. Examples ofcomplementary sequences include coding and non-coding sequences, whereinthe non-coding sequence contains complementary nucleotides to the codingsequence and thus forms the complement of the coding sequence. A furtherexample of complementary sequences are sense and antisense sequences,wherein the sense sequence contains complementary nucleotides to theantisense sequence and thus forms the complement of the antisensesequence. “Duplex” means at least two oligonucleotides and/orpolynucleotides that are fully or partially complementary undergoWatson-Crick type base pairing among all or most of their nucleotides sothat a stable complex is formed.

As described herein, the complementarity of sequences may be partial, inwhich only some of the nucleic acids match according to base pairing, orcomplete, where all the nucleic acids match according to base pairing.Thus, two sequences that are complementary to each other, may have aspecified percentage of nucleotides that complement one another (e.g.,about 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or higher complementarity over a specifiedregion). In embodiments, two sequences are complementary when they arecompletely complementary, having 100% complementarity. In embodiments,sequences in a pair of complementary sequences form portions of a singlepolynucleotide with non-base-pairing nucleotides (e.g., as in a hairpinor loop structure, with or without an overhang) or portions of separatepolynucleotides. In embodiments, one or both sequences in a pair ofcomplementary sequences form portions of longer polynucleotides, whichmay or may not include additional regions of complementarity.

The term “cleavable linker” or “cleavable moiety” as used herein refersto a divalent or monovalent, respectively, moiety which is capable ofbeing separated (e.g., detached, split, disconnected, hydrolyzed, astable bond within the moiety is broken) into distinct entities. Inembodiments, a cleavable linker is cleavable (e.g., specificallycleavable) in response to external stimuli (e.g., enzymes,nucleophilic/basic reagents, reducing agents, photo-irradiation,electrophilic/acidic reagents, organometallic and metal reagents, oroxidizing reagents). In embodiments, a cleavable linker is aself-immolative linker, a trivalent linker, or a linker capable ofdendritic amplication of signal, or a self-immolative dendrimercontaining linker (e.g., all as described in US 2007/0009980, US2006/0003383, and US 2009/0047699, which are incorporated by referencein their entirety for any purpose). A chemically cleavable linker refersto a linker which is capable of being split in response to the presenceof a chemical (e.g., acid, base, oxidizing agent, reducing agent, Pd(0),tris-(2-carboxyethyl)phosphine, dilute nitrous acid, fluoride,tris(3-hydroxypropyl)phosphine), sodium dithionite (Na₂S₂O₄), hydrazine(N₂H₄)). A chemically cleavable linker is non-enzymatically cleavable.In embodiments, the cleavable linker is cleaved by contacting thecleavable linker with a cleaving agent. In embodiments, the cleavingagent is sodium dithionite (Na₂S₂O₄), weak acid, hydrazine (N₂H₄),Pd(0), or light-irradiation (e.g., ultraviolet radiation). Inembodiments, cleaving includes removing. A “cleavable site” or “scissilelinkage” in the context of a polynucleotide is a site which allowscontrolled cleavage of the polynucleotide strand (e.g., the linker, theprimer, or the polynucleotide) by chemical, enzymatic, or photochemicalmeans known in the art and described herein. A scissile site may referto the linkage of a nucleotide between two other nucleotides in anucleotide strand (i.e., an internucleosidic linkage). In embodiments,the scissile linkage can be located at any position within the one ormore nucleic acid molecules, including at or near a terminal end (e.g.,the 3′ end of an oligonucleotide) or in an interior portion of the oneor more nucleic acid molecules. In embodiments, conditions suitable forseparating a scissile linkage include a modulating the pH and/or thetemperature. In embodiments, a scissile site can include at least oneacid-labile linkage. For example, an acid-labile linkage may include aphosphoramidate linkage. In embodiments, a phosphoramidate linkage canbe hydrolysable under acidic conditions, including mild acidicconditions such as trifluoroacetic acid and a suitable temperature(e.g., 30° C.), or other conditions known in the art, for exampleMatthias Mag, et al Tetrahedron Letters, Volume 33, Issue 48, 1992,7319-7322. In embodiments, the scissile site can include at least onephotolabile internucleosidic linkage (e.g., o-nitrobenzyl linkages, asdescribed in Walker et al, J. Am. Chem. Soc. 1988, 110, 21, 7170-7177),such as o-nitrobenzyloxymethyl or p-nitrobenzyloxymethyl group(s). Inembodiments, the scissile site includes at least one uracil nucleobase.In embodiments, a uracil nucleobase can be cleaved with a uracil DNAglycosylase (UDG) or Formamidopyrimidine DNA Glycosylase Fpg. Inembodiments, the scissile linkage site includes a sequence-specificnicking site having a nucleotide sequence that is recognized and nickedby a nicking endonuclease enzyme or a uracil DNA glycosylase. The term“self-immolative” referring to a linker is used in accordance with itswell understood meaning in Chemistry and Biology as used in US2007/0009980, US 2006/0003383, and US 2009/0047699, which areincorporated by reference in their entirety for any purpose. Inembodiments “self-immolative” referring to a linker refers to a linkerthat is capable of additional cleavage following initial cleavage by anexternal stimuli. The term dendrimer is used in accordance with its wellunderstood meaning in Chemistry. In embodiments, the term“self-immolative dendrimer” is used as described in US 2007/0009980, US2006/0003383, and US 2009/0047699, which are incorporated by referencein their entirety for any purpose and in embodiments refers to adendrimer that is capable of releasing all of its tail units through aself-immolative fragmentation following initial cleavage by an externalstimulus.

A “photocleavable linker” (e.g., including or consisting of ano-nitrobenzyl group) refers to a linker which is capable of being splitin response to photo-irradiation (e.g., ultraviolet radiation). Anacid-cleavable linker refers to a linker which is capable of being splitin response to a change in the pH (e.g., increased acidity). Abase-cleavable linker refers to a linker which is capable of being splitin response to a change in the pH (e.g., decreased acidity). Anoxidant-cleavable linker refers to a linker which is capable of beingsplit in response to the presence of an oxidizing agent. Areductant-cleavable linker refers to a linker which is capable of beingsplit in response to the presence of a reducing agent (e.g.,tris(3-hydroxypropyl)phosphine). In embodiments, the cleavable linker isa dialkylketal linker (Binaulda S., et al., Chem. Commun., 2013, 49,2082-2102; Shenoi R. A., et al., J. Am. Chem. Soc., 2012, 134,14945-14957), an azo linker (Rathod, K. M., et al., Chem. Sci. Tran.,2013, 2, 25-28; Leriche G., et al., Eur. J. Org. Chem., 2010, 23,4360-64), an allyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

The term “orthogonally cleavable linker” or “orthogonal cleavablelinker” as used herein refer to a cleavable linker that is cleaved by afirst cleaving agent (e.g., enzyme, nucleophilic/basic reagent, reducingagent, photo-irradiation, electrophilic/acidic reagent, organometallicand metal reagent, oxidizing reagent) in a mixture of two or moredifferent cleaving agents and is not cleaved by any other differentcleaving agent in the mixture of two or more cleaving agents. Forexample, two different cleavable linkers are both orthogonal cleavablelinkers when a mixture of the two different cleavable linkers arereacted with two different cleaving agents and each cleavable linker iscleaved by only one of the cleaving agents and not the other cleavingagent and the agent that cleaves each cleavable linker is different. Inembodiments, an orthogonally is a cleavable linker that followingcleavage the two separated entities (e.g., fluorescent dye, bioconjugatereactive group) do not further react and form a new orthogonallycleavable linker.

The term “orthogonal detectable label” or “orthogonal detectable moiety”as used herein refer to a detectable label (e.g., fluorescent dye ordetectable dye) that is capable of being detected and identified (e.g.,by use of a detection means (e.g., emission wavelength, physicalcharacteristic measurement)) in a mixture or a panel (collection ofseparate samples) of two or more different detectable labels. Forexample, two different detectable labels that are fluorescent dyes areboth orthogonal detectable labels when a panel of the two differentfluorescent dyes is subjected to a wavelength of light that is absorbedby one fluorescent dye but not the other and results in emission oflight from the fluorescent dye that absorbed the light but not the otherfluorescent dye. Orthogonal detectable labels may be separatelyidentified by different absorbance or emission intensities of theorthogonal detectable labels compared to each other and not only be theabsolute presence of absence of a signal. An example of a set of fourorthogonal detectable labels is the set of Rox-Labeled Tetrazine,Alexa488-Labeled SHA, Cy5-Labeled Streptavidin, and R6G-LabeledDibenzocyclooctyne.

The term “polymerase-compatible cleavable moiety” or “reversibleterminator” as used herein refers to a cleavable moiety which does notinterfere with a function of a polymerase (e.g., DNA polymerase,modified DNA polymerase, in incorporating the nucleotide, to which thepolymerase-compatible cleavable moiety is attached, to the 3′ end of thenewly formed nucleotide strand). Methods for determining the function ofa polymerase contemplated herein are described in B. Rosenblum et al.(Nucleic Acids Res. 1997 Nov. 15; 25(22): 4500-4504); and Z. Zhu et al.(Nucleic Acids Res. 1994 Aug. 25; 22(16): 3418-3422), which areincorporated by reference herein in their entirety for all purposes. Inembodiments, the polymerase-compatible cleavable moiety does notdecrease the function of a polymerase relative to the absence of thepolymerase-compatible cleavable moiety. In embodiments, thepolymerase-compatible cleavable moiety does not negatively affect DNApolymerase recognition. In embodiments, the polymerase-compatiblecleavable moiety does not negatively affect (e.g., limit) the readlength of the DNA polymerase. Additional examples of apolymerase-compatible cleavable moiety may be found in U.S. Pat. No.6,664,079, Ju J. et al. (2006) Proc Natl Acad Sci USA103(52):19635-19640; Ruparel H. et al. (2005) Proc Natl Acad Sci USA102(17):5932-5937; Wu J. et al. (2007) Proc Natl Acad Sci USA104(104):16462-16467; Guo J. et al. (2008) Proc Natl Acad Sci USA105(27): 9145-9150 Bentley D. R. et al. (2008) Nature 456(7218):53-59;or Hutter D. et al. (2010) Nucleosides Nucleotides & Nucleic Acids29:879-895, which are incorporated herein by reference in their entiretyfor all purposes. In embodiments, a polymerase-compatible cleavablemoiety includes an azido moiety or a dithiol linking moiety. Inembodiments, the polymerase-compatible cleavable moiety is —NH₂, —CN,—CH₃, C₂-C₆ allyl (e.g., —CH₂—CH═CH₂), methoxyalkyl (e.g., —CH₂—O—CH₃),or —CH₂N₃. In embodiments, the polymerase-compatible cleavable moietyincludes an ester (O—C(O)R^(Z)′ wherein R^(Z)′ is any alkyl or arylgroup which can include a formate, benzoyl formate, acetate, substitutedacetate, propionate, and other esters as described in Green, T. W.(Protective Groups in Organic Chemistry, Wiley & Sons, New York, 1981)).In embodiments, the polymerase-compatible cleavable moiety includes anether (O—R^(ZZ) wherein R^(ZZ) can be substituted or unsubstituted alkylsuch as methyl, substituted methyl, ethyl, substituted ethyl, allyl,substituted benzyl, silyl, or any other ether used to transientlyprotect hydroxyls and similar groups). In embodiments, thepolymerase-compatible cleavable moiety includes an O—CH₂(OC₂H₅)_(M)CH3wherein M is an integer from 1-10. In embodiments, thepolymerase-compatible cleavable moiety includes a phosphate,phosphoramidate, phosphoramide, toluic acid ester, benzoic ester, aceticacid ester, or ethoxyethyl ether. In embodiments, thepolymerase-compatible cleavable moiety comprises a disulfide moiety. Inembodiments, a polymerase-compatible cleavable moiety is a cleavablemoiety on a nucleotide, nucleobase, nucleoside, or nucleic acid thatdoes not interfere with a function of a polymerase (e.g., DNApolymerase, modified DNA polymerase).

The term “allyl” as described herein refers to an unsubstitutedmethylene attached to a vinyl group (i.e., —CH═CH₂), having the formula

An “allyl linker” refers to a divalent unsubstituted methylene attachedto a vinyl group, having the formula

The term “polymerase-compatible moiety” as used herein refers a moietywhich does not interfere with the function of a polymerase (e.g., DNApolymerase, modified DNA polymerase) in incorporating the nucleotide towhich the polymerase-compatible moiety is attached to the 3′ end of thenewly formed nucleotide strand. The polymerase-compatible moiety does,however, interfere with the polymerase function by preventing theaddition of another nucleotide to the 3′ oxygen of the nucleotide towhich the polymerase-compatible moiety is attached. Methods fordetermining the function of a polymerase contemplated herein aredescribed in B. Rosenblum et al. (Nucleic Acids Res. 1997 Nov. 15;25(22): 4500-4504); and Z. Zhu et al. (Nucleic Acids Res. 1994 Aug. 25;22(16): 3418-3422), which are incorporated by reference herein in theirentirety for all purposes. In embodiments the polymerase-compatiblemoiety does not decrease the function of a polymerase relative to theabsence of the polymerase-compatible moiety. In embodiments, thepolymerase-compatible moiety does not negatively affect DNA polymeraserecognition. In embodiments, the polymerase-compatible moiety does notnegatively affect (e.g., limit) the read length of the DNA polymerase.Additional examples of a polymerase-compatible moiety may be found inU.S. Pat. No. 6,664,079, Ju J. et al. (2006) Proc Natl Acad Sci USA103(52):19635-19640; Ruparel H. et al. (2005) Proc Natl Acad Sci USA102(17):5932-5937; Wu J. et al. (2007) Proc Natl Acad Sci USA104(104):16462-16467; Guo J. et al. (2008) Proc Natl Acad Sci USA105(27): 9145-9150 Bentley D. R. et al. (2008) Nature 456(7218):53-59;or Hutter D. et al. (2010) Nucleosides Nucleotides & Nucleic Acids29:879-895, which are incorporated herein by reference in their entiretyfor all purposes. In embodiments, a polymerase-compatible moietyincludes hydrogen, —N₃, —CN, or halogen. In embodiments, apolymerase-compatible moiety is a moiety on a nucleotide, nucleobase,nucleoside, or nucleic acid that does not interfere with the function ofa polymerase (e.g., DNA polymerase, modified DNA polymerase).

“Polymerase,” as used herein, refers to any natural or non-naturallyoccurring enzyme or other catalyst that is capable of catalyzing apolymerization reaction, such as the polymerization of nucleotidemonomers to form a nucleic acid polymer. Exemplary types of polymerasesthat may be used in the compositions and methods of the presentdisclosure include the nucleic acid polymerases such as DNA polymerase,DNA- or RNA-dependent RNA polymerase, and reverse transcriptase. In somecases, the DNA polymerase is 9° N polymerase or a variant thereof, E.Coli DNA polymerase I, Bacteriophage T4 DNA polymerase, Sequenase, TaqDNA polymerase, DNA polymerase from Bacillus stearothermophilus, Bst 2.0DNA polymerase, 9° N polymerase, 9° N polymerase (exo-)A485L/Y409V,Phi29 DNA Polymerase (φ29 DNA Polymerase), T7 DNA polymerase, DNApolymerase II, DNA polymerase III holoenzyme, DNA polymerase IV, DNApolymerase V, VentR DNA polymerase, Therminator™ II DNA Polymerase,Therminator™ III DNA Polymerase, or or Therminator™ IX DNA Polymerase.In embodiments, the polymerase is a protein polymerase.

As used herein, the term “DNA polymerase” and “nucleic acid polymerase”are used in accordance with their plain ordinary meanings and refer toenzymes capable of synthesizing nucleic acid molecules from nucleotides(e.g., deoxyribonucleotides). Typically, a DNA polymerase addsnucleotides to the 3′-end of a DNA strand, one nucleotide at a time. Inembodiments, the DNA polymerase is a Pol I DNA polymerase, Pol II DNApolymerase, Pol III DNA polymerase, Pol IV DNA polymerase, Pol V DNApolymerase, Pol β DNA polymerase, Pol μ DNA polymerase, Pol λ DNApolymerase, Pol σ DNA polymerase, Pol α DNA polymerase, Pol δ DNApolymerase, Pol ε DNA polymerase, Pol η DNA polymerase, Pol ι DNApolymerase, Pol κ DNA polymerase, Pol ζ DNA polymerase, Pol γ DNApolymerase, Pol θ DNA polymerase, Pol υ DNA polymerase, or athermophilic nucleic acid polymerase (e.g. Therminator γ, 9° Npolymerase (exo-), Therminator II, Therminator III, or Therminator IX).In embodiments, the DNA polymerase is a modified archaeal DNApolymerase. In embodiments, the polymerase is a reverse transcriptase.In embodiments, the polymerase is a mutant P. abyssi polymerase (e.g.,such as a mutant P. abyssi polymerase described in WO 2018/148723 or WO2020/056044).

As used herein, the term “thermophilic nucleic acid polymerase” refersto a family of DNA polymerases (e.g., 9° N™) and mutants thereof derivedfrom the DNA polymerase originally isolated from the hyperthermophilicarchaea, Thermococcus sp. 9 degrees N-7, found in hydrothermal vents atthat latitude (East Pacific Rise) (Southworth M W, et al. PNAS. 1996;93(11):5281-5285). A thermophilic nucleic acid polymerase is a member ofthe family B DNA polymerases. Site-directed mutagenesis of the 3′-5′ exomotif I (Asp-Ile-Glu or DIE) to AIA, AIE, EIE, EID or DIA yieldedpolymerase with no detectable 3′ exonuclease activity. Mutation toAsp-Ile-Asp (DID) resulted in reduction of 3′-5′ exonuclease specificactivity to <1% of wild type, while maintaining other properties of thepolymerase including its high strand displacement activity. The sequenceAIA (D141A, E143A) was chosen for reducing exonuclease. Subsequentmutagenesis of key amino acids results in an increased ability of theenzyme to incorporate dideoxynucleotides, ribonucleotides andacyclonucleotides (e.g., Therminator II enzyme from New England Biolabswith D141A/E143A/Y409V/A485L mutations); 3′-amino-dNTPs, 3′-azido-dNTPsand other 3′-modified nucleotides (e.g., NEB Therminator III DNAPolymerase with D141A/E143A/L4085/Y409A/P410V mutations, NEB TherminatorIX DNA polymerase), or γ-phosphate labeled nucleotides (e.g.,Therminator γ:D141A/E143A/W355A/L408W/R460A/Q4615/K464E/D480V/R484W/A485L). Typically,these enzymes do not have 5′-3′ exonuclease activity. Additionalinformation about thermophilic nucleic acid polymerases may be found in(Southworth M W, et al. PNAS. 1996; 93(11):5281-5285; Bergen K, et al.ChemBioChem. 2013; 14(9):1058-1062; Kumar S, et al. Scientific Reports.2012; 2:684; Fuller C W, et al. 2016; 113(19):5233-5238; Guo J, et al.Proceedings of the National Academy of Sciences of the United States ofAmerica. 2008; 105(27):9145-9150), which are incorporated herein intheir entirety for all purposes.

As used herein, the term “exonuclease activity” is used in accordancewith its ordinary meaning in the art, and refers to the removal of anucleotide from a nucleic acid by a DNA polymerase. For example, duringpolymerization, nucleotides are added to the 3′ end of the primerstrand. Occasionally a DNA polymerase incorporates an incorrectnucleotide to the 3′-OH terminus of the primer strand, wherein theincorrect nucleotide cannot form a hydrogen bond to the correspondingbase in the template strand. Such a nucleotide, added in error, isremoved from the primer as a result of the 3′ to 5′ exonuclease activityof the DNA polymerase. In embodiments, exonuclease activity may bereferred to as “proofreading.” When referring to 3′-5′ exonucleaseactivity, it is understood that the DNA polymerase facilitates ahydrolyzing reaction that breaks phosphodiester bonds at the 3′ end of apolynucleotide chain to excise the nucleotide. In embodiments, 3′-5′exonuclease activity refers to the successive removal of nucleotides insingle-stranded DNA in a 3′→5′ direction, releasing deoxyribonucleoside5′-monophosphates one after another. Methods for quantifying exonucleaseactivity are known in the art, see for example Southworth et al, PNASVol 93, 8281-8285 (1996).

As used herein, the terms “polynucleotide primer” and “primer” refers toany polynucleotide molecule that may hybridize to a polynucleotidetemplate, be bound by a polymerase, and be extended in atemplate-directed process for nucleic acid synthesis. The primer may bea separate polynucleotide from the polynucleotide template, or both maybe portions of the same polynucleotide (e.g., as in a hairpin structurehaving a 3′ end that is extended along another portion of thepolynucleotide to extend a double-stranded portion of the hairpin).Primers (e.g., forward or reverse primers) may be attached to a solidsupport. A primer can be of any length depending on the particulartechnique it will be used for. For example, PCR primers are generallybetween 10 and 40 nucleotides in length. The length and complexity ofthe nucleic acid fixed onto the nucleic acid template may vary. In someembodiments, a primer has a length of 200 nucleotides or less. Incertain embodiments, a primer has a length of 10 to 150 nucleotides, 15to 150 nucleotides, 5 to 100 nucleotides, 5 to 50 nucleotides or 10 to50 nucleotides. One of skill can adjust these factors to provide optimumhybridization and signal production for a given hybridization procedure.The primer permits the addition of a nucleotide residue thereto, oroligonucleotide or polynucleotide synthesis therefrom, under suitableconditions. In an embodiment the primer is a DNA primer, i.e., a primerconsisting of, or largely consisting of, deoxyribonucleotide residues.The primers are designed to have a sequence that is the complement of aregion of template/target DNA to which the primer hybridizes. Theaddition of a nucleotide residue to the 3′ end of a primer by formationof a phosphodiester bond results in a DNA extension product. Theaddition of a nucleotide residue to the 3′ end of the DNA extensionproduct by formation of a phosphodiester bond results in a further DNAextension product. In another embodiment, the primer is an RNA primer.In embodiments, a primer is hybridized to a target polynucleotide. A“primer” is complementary to a polynucleotide template, and complexes byhydrogen bonding or hybridization with the template to give aprimer/template complex for initiation of synthesis by a polymerase,which is extended by the addition of covalently bonded bases linked atits 3′ end complementary to the template in the process of DNAsynthesis.

“Polymerase,” as used herein, refers to any natural or non-naturallyoccurring enzyme or other catalyst that is capable of catalyzing apolymerization reaction, such as the polymerization of nucleotidemonomers to form a nucleic acid polymer. Exemplary types of polymerasesthat may be used in the compositions and methods of the presentdisclosure include the nucleic acid polymerases such as DNA polymerase,DNA- or RNA-dependent RNA polymerase, and reverse transcriptase. In somecases, the DNA polymerase is 9° N polymerase or a variant thereof, E.Coli DNA polymerase I, Bacteriophage T4 DNA polymerase, Sequenase, TaqDNA polymerase, DNA polymerase from Bacillus stearothermophilus, Bst 2.0DNA polymerase, 9° N polymerase, 9° N polymerase (exo-)A485L/Y409V,Phi29 DNA Polymerase (φ29 DNA Polymerase), T7 DNA polymerase, DNApolymerase II, DNA polymerase III holoenzyme, DNA polymerase IV, DNApolymerase V, VentR DNA polymerase, Therminator™ II DNA Polymerase,Therminator™ III DNA Polymerase, or or Therminator™ IX DNA Polymerase.In embodiments, the polymerase is a protein polymerase.

The phrase “stringent hybridization conditions” refers to conditionsunder which a primer will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C.

The term “polymer” refers to a molecule including repeating subunits(e.g., polymerized monomers). For example, polymeric molecules may bebased upon polyethylene glycol (PEG), tetraethylene glycol (TEG),polyvinylpyrrolidone (PVP), poly(xylene), or poly(p-xylylene). The term“polymerizable monomer” is used in accordance with its meaning in theart of polymer chemistry and refers to a compound that may covalentlybind chemically to other monomer molecules (such as other polymerizablemonomers that are the same or different) to form a polymer.

“Solid substrate” shall mean any suitable medium present in the solidphase to which a nucleic acid or an agent may be affixed. Non-limitingexamples include chips, beads and columns. The solid substrate can benon-porous or porous. Exemplary solid substrates include, but are notlimited to, glass and modified or functionalized glass, plastics(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyurethanes,Teflon™, cyclic olefins, polyimides, etc.), nylon, ceramics, resins,Zeonor, silica or silica-based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses, optical fiber bundles, andpolymers. In embodiments, the solid substrate for have at least onesurface located within a flow cell. The solid substrate, or regionsthereof, can be substantially flat. The solid substrate can have surfacefeatures such as wells, pits, channels, ridges, raised regions, pegs,posts or the like. The term solid substrate is encompassing of asubstrate (e.g., a flow cell) having a surface comprising a polymercoating covalently attached thereto. In embodiments, the solid substrateis a flow cell. The term “flowcell” or “flow cell” as used herein refersto a chamber including a solid surface across which one or more fluidreagents can be flowed. Examples of flowcells and related fluidicsystems and detection platforms that can be readily used in the methodsof the present disclosure are described, for example, in Bentley et al.,Nature 456:53-59 (2008).

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous references, e.g.,Current Protocols in Molecular Biology, ed. Ausubel, et al., supra.

Where a range of values is provided herein, it is understood that eachintervening value, to the tenth of the unit (if appropriate) of thelower limit unless the context clearly dictates otherwise, between theupper and lower limit of that range, and any other stated or interveningvalue in that stated range, is encompassed within the invention. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges, and are also encompassed within theinvention, subject to any specifically excluded limit in the statedrange. Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe invention.

While various embodiments of the invention are shown and describedherein, it will be understood by those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutes may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

The term “protecting group” is used in accordance with its ordinarymeaning in organic chemistry and refers to a moiety covalently bound toa heteroatom, heterocycloalkyl, or heteroaryl to prevent reactivity ofthe heteroatom, heterocycloalkyl, or heteroaryl during one or morechemical reactions performed prior to removal of the protecting group.Typically a protecting group is bound to a heteroatom (e.g., O) during apart of a multipart synthesis wherein it is not desired to have theheteroatom react (e.g., a chemical reduction) with the reagent.Following protection the protecting group may be removed (e.g., bymodulating the pH). In embodiments, the protecting group is an alcoholprotecting group. Non-limiting examples of alcohol protecting groupsinclude acetyl, benzoyl, benzyl, methoxymethyl ether (MOM),tetrahydropyranyl (THP), and silyl ether (e.g., trimethylsilyl (TMS)).In embodiments, the protecting group is an amine protecting group.Non-limiting examples of amine protecting groups include carbobenzyloxy(Cbz), tert-butyloxycarbonyl (BOC), 9-Fluorenylmethyloxycarbonyl (FMOC),acetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl ether (PMB), andtosyl (Ts). In embodiments, the protecting group is a nucleosideprotecting group. In embodiments, the protecting group is a5′-O-nucleoside protecting group.

The term “5′-nucleoside protecting group” as used herein refers to amoiety covalently bound to a heteroatom (e.g., O) on the 5′ position ofsugar to prevent reactivity of the heteroatom during one or morechemical reactions performed prior to removal of the protecting group.Typically a protecting group is bound to a heteroatom (e.g., 0) during apart of a multipart synthesis wherein it is not desired to have theheteroatom react (e.g., during a chemical reduction) with the reagent.Following protection the protecting group may be removed by anyappropriate means (e.g., by modulating the pH). Non-limiting examples of5′-O-nucleoside protecting groups include silyl ethers (e.g.,tert-butyl-diphenylsilyl (TBDPS), or primary and secondarytert-butyldimethylsilyl (TBDMS)) or trityl (e.g., 4,4′-dimethoxytrityl(DMT)). In embodiments, R¹ includes a protecting group found in Green'sProtective Groups in Organic Chemistry, Wiley, Fourth edition, 2007,Peter G. M. Wuts and Theodora W. Greene, and Current Protocols inNucleic Acid Chemistry (2000) 2.3.1-2.3.34, John Wiley & Sons, Inc.,which is incorporated herein by reference in its entirety for allpurposes. The terms “5′-nucleoside protecting group” and“5′-O-nucleoside protecting group” are used interchangeably herein.

The term “deprotect” or “deprotecting” is used in accordance with itsordinary meaning in organic chemistry and refers a process or chemicalreaction that remove a protecting group, which is covalently bound to aheteroatom, heterocycloalkyl, or heteroaryl, to recover reactivity ofthe heteroatom, heterocycloalkyl, or heteroaryl for subsequent chemicalreactions or metabolic pathway. The term “deprotecting agent” or“deprotecting reagent” is used in accordance with its ordinary meaningin organic chemistry and refers to a molecule used for deprotecting. Inembodiments, the deprotecting agent is an acid or a base. Inembodiments, the deprotecting agent includes alpha-hydroxy amines (aminoalcohol), primary amines and secondary amines. In embodiments, thedeprotecting agent is ammonium salt (e.g., ammonium hydroxide, ammoniumhydrogen sulfate, ceric ammonium nitrate, or ammonium fluoride). Inembodiments, the deprotecting agent is concentrated ammonium hydroxide.

The term “reaction vessel” is used in accordance with its ordinarymeaning in chemistry or chemical engineering and refers to a containerhaving an inner volume in which a reaction takes place. In embodiments,the reaction vessel may be designed to provide suitable reactionconditions such as reaction volume, reaction temperature or pressure,and stirring or agitation, which may be adjusted to ensure that thereaction proceeds with a desired, sufficient or highest efficiency forproducing a product from the chemical reaction. In embodiments, thereaction vessel is a container for liquid, gas or solid. In embodiments,the reaction vessel may include an inlet, an outlet, a reservoir and thelike. In embodiments, the reaction vessel is connected to a pump (e.g.,vacuum pump), a controller (e.g., CPU), or a monitoring device (e.g., UVdetector or spectrophotometer). In embodiments, the reaction vessel is aflow cell. In embodiments, the reaction vessel is within a sequencingdevice.

A person of ordinary skill in the art will understand when a variable(e.g., moiety or linker) of a compound or of a compound genus (e.g., agenus described herein) is described by a name or formula of astandalone compound with all valencies filled, the unfilled valence(s)of the variable will be dictated by the context in which the variable isused. For example, when a variable of a compound as described herein isconnected (e.g., bonded) to the remainder of the compound through asingle bond, that variable is understood to represent a monovalent form(i.e., capable of forming a single bond due to an unfilled valence) of astandalone compound (e.g., if the variable is named “methane” in anembodiment but the variable is known to be attached by a single bond tothe remainder of the compound, a person of ordinary skill in the artwould understand that the variable is actually a monovalent form ofmethane, i.e., methyl or —CH₃). Likewise, for a linker variable (e.g.,L¹, L², or L³ as described herein), a person of ordinary skill in theart will understand that the variable is the divalent form of astandalone compound (e.g., if the variable is assigned to “PEG” or“polyethylene glycol” in an embodiment but the variable is connected bytwo separate bonds to the remainder of the compound, a person ofordinary skill in the art would understand that the variable is adivalent (i.e., capable of forming two bonds through two unfilledvalences) form of PEG instead of the standalone compound PEG).

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of reaction assays, such deliverysystems include systems that allow for the storage, transport, ordelivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. inthe appropriate containers) and/or supporting materials (e.g.,packaging, buffers, written instructions for performing a method, etc.)from one location to another. For example, kits include one or moreenclosures (e.g., boxes) containing the relevant reaction reagentsand/or supporting materials. As used herein, the term “fragmented kit”refers to a delivery system comprising two or more separate containersthat each contain a subportion of the total kit components. Thecontainers may be delivered to the intended recipient together orseparately. For example, a first container may contain an enzyme for usein an assay, while a second container contains oligonucleotides. Incontrast, a “combined kit” refers to a delivery system containing all ofthe components of a reaction assay in a single container (e.g., in asingle box housing each of the desired components). The term “kit”includes both fragmented and combined kits.

As used herein, the terms “sequencing”, “sequence determination”,“determining a nucleotide sequence”, and the like include determinationof a partial or complete sequence information, including theidentification, ordering, or locations of the nucleotides that comprisethe polynucleotide being sequenced, and inclusive of the physicalprocesses for generating such sequence information. That is, the termincludes sequence comparisons, consensus sequence determination, contigassembly, fingerprinting, and like levels of information about a targetpolynucleotide, as well as the express identification and ordering ofnucleotides in a target polynucleotide. The term also includes thedetermination of the identification, ordering, and locations of one,two, or three of the four types of nucleotides within a targetpolynucleotide. In some embodiments, a sequencing process describedherein comprises contacting a template and an annealed primer with asuitable polymerase under conditions suitable for polymerase extensionand/or sequencing. The sequencing methods are preferably carried outwith the target polynucleotide arrayed on a solid substrate. Multipletarget polynucleotides can be immobilized on the solid support throughlinker molecules, or can be attached to particles, e.g., microspheres,which can also be attached to a solid substrate. In embodiments, thesolid substrate is in the form of a chip, a bead, a well, a capillarytube, a slide, a wafer, a filter, a fiber, a porous media, or a column.In embodiments, the solid substrate is gold, quartz, silica, plastic,glass, diamond, silver, metal, or polypropylene. In embodiments, thesolid substrate is porous.

As used herein, the term “extension” or “elongation” is used inaccordance with its plain and ordinary meanings and refer to synthesisby a polymerase of a new polynucleotide strand complementary to atemplate strand by adding free nucleotides (e.g., dNTPs) from a reactionmixture that are complementary to the template in the 5′-to-3′direction. Extension includes condensing the 5′-phosphate group of thedNTPs with the 3′-hydroxy group at the end of the nascent (elongating)polynucleotide strand.

As used herein, the term “sequencing read” is used in accordance withits plain and ordinary meaning and refers to an inferred sequence ofnucleotide bases (or nucleotide base probabilities) corresponding to allor part of a single polynucleotide fragment. A sequencing read mayinclude 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or morenucleotide bases. In embodiments, a sequencing read includes reading abarcode sequence and a template nucleotide sequence. In embodiments, asequencing read includes reading a template nucleotide sequence. Inembodiments, a sequencing read includes reading a barcode and not atemplate nucleotide sequence.

II. Compounds, Compositions & Kits

In an aspect is provided a compound having the formula:

B is a divalent nucleobase. R¹ is hydrogen, halogen, —CCl₃, —CBr₃, —CF₃,—CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,5′-nucleoside protecting group, 5′-O-nucleoside protecting group,monophosphate moiety, polyphosphate moiety, or nucleic acid moiety. R²and R³ are independently hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃,—CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl, apolymerase-compatible cleavable moiety, or a —O-polymerase-compatiblecleavable moiety. R⁴ is a detectable moiety. L¹⁰⁰ is a divalent linkerincluding

R⁵ and R⁶ are independently hydrogen, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. R⁷, R⁸, and R⁹ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.

In embodiments, the compound has the formula:

B is a divalent nucleobase. R¹ is a 5′-nucleoside protecting group,5′-O-nucleoside protecting group, monophosphate moiety, polyphosphatemoiety, or nucleic acid moiety. R² and R³ are independently hydrogen,halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl,—CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCl₃, —OCHCl₂,—OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, a polymerase-compatiblecleavable moiety, or a —O-polymerase-compatible cleavable moiety. R⁴ isa detectable moiety. L¹⁰⁰ is a divalent linker including

R⁵ and R⁶ are independently hydrogen, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. R⁷, R⁸, and R⁹ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl.

In another aspect is provided a compound having the formula

B is a divalent nucleobase. R¹ is hydrogen, halogen, —CCl₃, —CBr₃, —CF₃,—CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,5′-nucleoside protecting group, 5′-O-nucleoside protecting group,monophosphate moiety, polyphosphate moiety, or nucleic acid moiety. R²is hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂,—CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,—OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I,—OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, or apolymerase-compatible cleavable moiety. R³ is a —O-polymerase compatiblecleavable moiety having the formula:

R⁴ is a detectable moiety. L¹⁰⁰ is a divalent linker. R⁵ and R⁶ areindependently hydrogen, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. R⁷,R⁸, and R⁹ are independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. R¹⁰ is substituted or unsubstituted alkyl.

In embodiments, the compound has the formula

B is a divalent nucleobase. R¹ is a 5′-nucleoside protecting group,5′-O-nucleoside protecting group, monophosphate moiety, polyphosphatemoiety, or nucleic acid moiety. R² is hydrogen, halogen, —CCl₃, —CBr₃,—CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl, or apolymerase-compatible cleavable moiety. R³ is a —O-polymerase compatiblecleavable moiety having the formula:

R⁴ is a detectable moiety. L¹⁰⁰ is a divalent linker. R⁵ and R⁶ areindependently hydrogen, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. R⁷,R⁸, and R⁹ are independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. R¹⁰ is substituted or unsubstituted alkyl.

In embodiments, the compounds of Formula (I′), Formula (I), Formula(II′), and/or Formula (II) are referred to as nucleotides, modifiednucleotides, or nucleotide analogues. In embodiments, the compounds ofFormula I′ include a nucleotide portion and a 3′-O-reversibleterminator. In embodiments, the compounds of Formula I include anucleotide portion and a 3′-O-reversible terminator. For example, thenucleotide portion is

and the 3′-O-reversible terminator portion is R³, as described herein.

In embodiments, R¹ is —OH, a 5′-nucleoside protecting group, a5′-O-nucleoside protecting group, monophosphate moiety, polyphosphatemoiety, or nucleic acid moiety. In embodiments, R¹ is a triphosphatemoiety. In embodiments, R¹ is —OH. In embodiments, R¹ is a5′-O-nucleoside protecting group. In embodiments, R¹ is a nucleic acidmoiety. In embodiments, le is independently a monophosphate moiety or aderivative thereof (e.g., including a phosphoramidate moiety,phosphorothioate moiety, phosphorodithioate moiety, orO-methylphosphoroamidite moiety), polyphosphate moiety or derivativethereof (e.g., including a phosphoramidate, phosphorothioate,phosphorodithioate, or O-methylphosphoroamidite), or nucleic acid moietyor derivative thereof (e.g., including a phosphoramidate,phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite). Inembodiments, R¹ is a 5′-nucleoside protecting group. In embodiments, R¹is a 5′-O-nucleoside protecting group. In embodiments, the 5′-nucleosideprotecting group is a protecting group attached to the 5′ carbon of thenucleoside. In embodiments, the 5′-O-nucleoside protecting group is aprotecting group attached to the hydroxyl group of the 5′ carbon of thenucleoside.

In embodiments, R¹ is hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃,—CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl, a5′-O-nucleoside protecting group, monophosphate moiety, polyphosphatemoiety, or nucleic acid moiety. In embodiments, R¹ is a monophosphatemoiety including a phosphodiester derivative. In embodiments, R¹ is apolyphosphate moiety including a phosphodiester derivative. Inembodiments, R¹ is a nucleic acid moiety including a phosphodiesterderivative. In embodiments, R¹ is a phosphoramidate moiety. Inembodiments, R¹ is a polyphosphate moiety including a phosphoramidate.In embodiments, R¹ is a nucleic acid moiety including a phosphoramidate.In embodiments, R¹ is a phosphorothioate moiety. In embodiments, R¹ is apolyphosphate moiety including a phosphorothioate. In embodiments, R¹ isa nucleic acid moiety including a phosphorothioate. In embodiments, R¹is a phosphorodithioate moiety. In embodiments, R¹ is a polyphosphatemoiety including a phosphorodithioate. In embodiments, R¹ is a nucleicacid moiety including a phosphorodithioate. In embodiments, R¹ is anO-methylphosphoroamidite moiety. In embodiments, R¹ is a polyphosphatemoiety including an O-methylphosphoroamidite. In embodiments, R¹ is anucleic acid moiety including an O-methylphosphoroamidite. Inembodiments, R¹ is a nucleic acid moiety including a nucleotide analog.In embodiments, R¹ is a nucleic acid moiety including a plurality ofoptionally different nucleotide analogs.

In embodiments, R¹ is a monophosphate moiety. In embodiments, R¹ is atriphosphate moiety. In embodiments, R¹ is a polyphosphate moiety. Inembodiments, R¹ is a nucleic acid moiety. In embodiments, R¹ has theformula:

or ionized forms thereof. In embodiments, R¹ has the formula

or ionized forms thereof. In embodiments, R¹ has the formula:

or ionized forms thereof. In embodiments, R¹ has the formula:

or ionized forms thereof, wherein np is an integer of 1 or greater. Inembodiments, np is an integer from 1 to 5. In embodiments, np is 1. Inembodiments, np is 2.

In embodiments, R¹ is a 5′-O-nucleoside protecting group, for example a5′ nucleoside protecting group known in the art include those describedin Seliger H. Curr. Protoc Nucleic Acid Chem. 2001; Chapter 2 or K. Seioet al, Nucleic Acids Research Supplement 2, 27-28 (2002); both of whichare incorporated by reference for all purposes. Non-limiting examples of5′-O-nucleoside protecting groups include 2,2,2-Trichloroethyl carbonate(Troc), 2-Methoxyethoxymethyl ether (MEM), 2-Naphthylmethyl ether (Nap),4-Methoxybenzyl ether (PMB), Acetate (Ac), Benzoate (Bz), Benzyl ether(Bn), Benzyloxymethyl acetal (BOM), Ethoxyethyl acetal (EE),Methoxymethyl acetal (MOM), Methoxypropyl acetal (MOP), Methyl ether,Tetrahydropyranyl acetal (THP), Triethylsilyl ether (TES),Triisopropylsilyl ether (TIPS), Trimethylsilyl ether (TMS),tert-Butyldimethylsilyl ether (TBS, TBDMS), or tert-butyldiphenylsilylether (TBDPS). In embodiments, R¹ is

In embodiments, R¹ is hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃,—CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₂NH₂, —NHNH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,—OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I,—OCH₂F, —N₃, —SF₅, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2to 8, 2 to 6, 4 to 6, 2 to 3, or 4 to 5 membered), substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted cycloalkyl (e.g., C₃-C₈,C₃-C₆, C₄-C₆, or C₅-C₆), substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, 4 to 6,4 to 5, or 5 to 6 membered), substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl (e.g., 5to 10 membered, 5 to 9 membered, or 5 to 6 membered), or a5′-O-nucleoside protecting group; or R¹ is a monophosphate moiety,polyphosphate moiety, or nucleic acid moiety. In embodiments, R¹ ishydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂,—CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₂NH₂, —NHNH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCl₃, —OCHCl₂,—OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl(e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heteroalkyl (e.g., 2 to 8, 2 to 6, 4to 6, 2 to 3, or 4 to 5 membered), substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, orC₅-C₆), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, 4 to 6, 4 to 5, or5 to 6 membered), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, R¹ is a5′-O-nucleoside protecting group. In embodiments, R¹ is a monophosphatemoiety, polyphosphate moiety, or nucleic acid moiety. In embodiments, R¹is a monophosphate moiety. In embodiments, R¹ is a polyphosphate moiety.In embodiments, R¹ is a nucleic acid moiety. In embodiments, R¹ ishydrogen. In embodiments, R¹ is a triphosphate moiety. In embodiments,R¹ is —OH.

In embodiments, a substituted R¹ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R¹ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R¹ is substituted, itis substituted with at least one substituent group. In embodiments, whenR¹ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R¹ is substituted, it issubstituted with at least one lower substituent group. In embodiments,when R¹ is substituted, it is substituted with 1 to 10 substituentgroups. In embodiments, when R¹ is substituted, it is substituted with 1to 10 size-limited substituent groups. In embodiments, when R¹ issubstituted, it is substituted with 1 to 10 lower substituent groups. Inembodiments, when R¹ is substituted, it is substituted with 1 to 5substituent groups. In embodiments, when R¹ is substituted, it issubstituted with 1 to 5 size-limited substituent groups. In embodiments,when R¹ is substituted, it is substituted with 1 to 5 lower substituentgroups. In embodiments, when R¹ is substituted, it is substituted with asubstituent group. In embodiments, when R¹ is substituted, it issubstituted with a size-limited substituent group. In embodiments, whenR¹ is substituted, it is substituted with a lower substituent group.

In embodiments, R¹ is —OH, a 5′-O-nucleoside protecting group,monophosphate moiety, polyphosphate moiety, or nucleic acid moiety. Inembodiments, R¹ is a polyphosphate moiety. In embodiments, R¹ is atriphosphate moiety.

In embodiments, R² is hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃,—CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl; or apolymerase-compatible cleavable moiety. In embodiments, R² is hydrogen.In embodiments, R² is —OH. In embodiments, R² is an—O-polymerase-compatible cleavable moiety, wherein the —O— is attachedto the 2′ position of the ribose sugar of a nucleotide and apolymerase-compatible cleavable moiety is as described herein.

In embodiments, R² is hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃,—CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, R^(2A)-substituted or unsubstitutedalkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),R^(2A)-substituted or unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20,2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), R^(2A)-substituted orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆),R^(2A)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to6, or 5 to 6 membered), R^(2A)-substituted or unsubstituted aryl (e.g.,C₆-C₁₀, C₁₀, or phenyl), or R^(2A)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments,R² is hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,—CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,—OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I,—OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl (e.g., C₁-C₂₀,C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆,or C₅-C₆), substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8,3 to 6, or 5 to 6 membered), substituted or unsubstituted aryl (e.g.,C₆-C₁₀, C₁₀, or phenyl), or substituted or unsubstituted heteroaryl(e.g., 5 to 10, 5 to 9, or 5 to 6 membered), polymerase-compatiblecleavable moiety; or a —O-polymerase-compatible cleavable moiety.

R^(2A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂T, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, R^(2B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(2B)-substituted or unsubstituted heteroalkyl(e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),R^(2B)-substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), R^(2B)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to8, 3 to 6, or 5 to 6 membered), R^(2B)-substituted or unsubstituted aryl(e.g., C₆-C₁₀, C₁₀, or phenyl), or R^(2B)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered), or apolymerase-compatible cleavable moiety. In embodiments, R^(2A) isindependently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,—CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,—OC₁₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I,—OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN, —ONO₂,R^(2B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈,C₁-C₆, or C₁-C₄), R^(2B)-substituted or unsubstituted heteroalkyl (e.g.,2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),R^(2B)-substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), R^(2B)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to8, 3 to 6, or 5 to 6 membered), R^(2B)-substituted or unsubstituted aryl(e.g., C₆-C₁₀, C₁₀, or phenyl), or R^(2B)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments,R^(2A) is independently a polymerase-compatible cleavable moiety.

R^(2B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, R^(2C)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(2C)-substituted or unsubstituted heteroalkyl(e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),R^(2C)-substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), R^(2C)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to8, 3 to 6, or 5 to 6 membered), R^(2C)-substituted or unsubstituted aryl(e.g., C₆-C₁₀, C₁₀, or phenyl), or R^(2C)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

R^(2C) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, orC₁-C₄), unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to8, 2 to 6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈,C₃-C₆, or C₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6,or 5 to 6 membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl),or unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, R² is hydrogen. In embodiments, R² is —OH. Inembodiments, R² is —O-polymerase-compatible cleavable moiety. Inembodiments, the -polymerase-compatible cleavable moiety is:

In embodiments, the -polymerase-compatible cleavable moiety is:

In embodiments, R² is H. In embodiments, R² is —OH.

In embodiments, R³ is hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃,—CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br,—OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl; or apolymerase-compatible cleavable moiety. In embodiments, R³ is hydrogen.In embodiments, R³ is —OH. In embodiments, R³ is an—O-polymerase-compatible cleavable moiety, wherein the —O— is attachedto the 3′ position of the ribose sugar of a nucleotide and apolymerase-compatible cleavable moiety is as described herein.

In embodiments, R³ is hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃,—CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br,—OCH₂I, —OCH₂F, —N₃, —SF₅, R^(3A)-substituted or unsubstituted alkyl(e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), R^(3A)-substituted orunsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), R^(3A)-substituted or unsubstituted cycloalkyl(e.g., C₃-C₈, C₃-C₆, or C₅-C₆), R^(3A)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(3A)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl),or R^(3A)-substituted or unsubstituted heteroaryl (e.g., 5 to 10, 5 to9, or 5 to 6 membered). In embodiments, R³ is hydrogen, halogen, —CCl₃,—CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F,—CH₂I, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂,—NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH,—NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂,—OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted orunsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),substituted or unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to10, 2 to 8, 2 to 6, or 2 to 4 membered), substituted or unsubstitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered), substitutedor unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), or substituted orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered); ora polymerase-compatible cleavable moiety.

R^(3A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, R^(3B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(3B)-substituted or unsubstituted heteroalkyl(e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),R^(3B)-substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), R^(3B)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to8, 3 to 6, or 5 to 6 membered), R^(3B)-substituted or unsubstituted aryl(e.g., C₆-C₁₀, C₁₀, or phenyl), or R^(3B)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered), or apolymerase-compatible cleavable moiety. In embodiments, R^(3A) isindependently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,—CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,—OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I,—OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN, —ONO₂,R^(3B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈,C₁-C₆, or C₁-C₄), R^(3B)-substituted or unsubstituted heteroalkyl (e.g.,2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),R^(3B)-substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, or C₅-C₆),R^(3B)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to6, or 5 to 6 membered), R^(3B)-substituted or unsubstituted aryl (e.g.,C₆-C₁₀, C₁₀, or phenyl), or R^(3B)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

R^(3B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CH₂Cl, —CH₂Br, —CH₂F, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,—OCl₃, —OCHCl₂, —OCHBr₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂F, —N₃, —SF₅,—NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN, —ONO₂, R^(3C)-substituted orunsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₆, or C₁-C₄),R^(3C)-substituted or unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20,2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), R^(3C)-substituted orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆),R^(3C)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to6, or 5 to 6 membered), R^(3C)-substituted or unsubstituted aryl (e.g.,C₆-C₁₀, C₁₀, or phenyl), or R^(3C)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

R^(3C) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,—OCl₃, —OCHCl₂, —OCHBr₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂F, —N₃, —SF₅,—NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN, —ONO₂, unsubstituted alkyl (e.g., C₁-C₂₀,C₁₀-C₂₀, C₁-C₈, C₁-C₆, or unsubstituted heteroalkyl (e.g., 2 to 20, 8 to20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), unsubstitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, R³ is hydrogen. In embodiments, R³ is —OH. Inembodiments, R³ is a —O-polymerase-compatible cleavable moiety whereinthe —O— is attached to the 3′ position of the ribose sugar of anucleotide and a polymerase-compatible cleavable moiety is as describedherein. In embodiments, the polymerase-compatible cleavable moiety is:

In embodiments, the polymerase-compatible cleavable moiety is:

In embodiments, R³ is a —O-reversible terminator moiety wherein the —O—is attached to the 3′ position of the ribose sugar of a nucleotide and areversible terminator moiety is as described herein. In embodiments, thereversible terminator moiety is

as described in U.S. Pat. No. 10,738,072, which is incorporated hereinby reference for all purposes. In embodiments, the reversible terminatormoiety is

In embodiments, the reversible terminator moiety is

In embodiments, B is a divalent nucleobase. In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is a cytosine or a derivative thereof, guanine or aderivative thereof, adenine or a derivative thereof, thymine or aderivative thereof, uracil or a derivative thereof, hypoxanthine or aderivative thereof, xanthine or a derivative thereof, 7-methylguanine ora derivative thereof, 5,6-dihydrouracil or a derivative thereof,5-methylcytosine or a derivative thereof, or 5-hydroxymethylcytosine ora derivative thereof. In embodiments, B is a substituted cytosine or aderivative thereof, substituted guanine or a derivative thereof,substituted adenine or a derivative thereof, substituted thymine or aderivative thereof, substituted uracil or a derivative thereof,substituted hypoxanthine or a derivative thereof, substituted xanthineor a derivative thereof, substituted 7-methylguanine or a derivativethereof, substituted 5,6-dihydrouracil or a derivative thereof,substituted 5-methylcytosine or a derivative thereof, or substituted5-hydroxymethylcytosine or a derivative thereof. In embodiments, B is asubstituted cytosine, substituted guanine, substituted adenine,substituted thymine, substituted uracil, substituted hypoxanthine,substituted xanthine, substituted 7-methylguanine, substituted5,6-dihydrouracil, substituted 5-methylcytosine, or a substituted5-hydroxymethylcytosine. In embodiments, B a substituted B issubstituted with at least one substituent group, size-limitedsubstituent group, or lower substituent group. In embodiments, when B issubstituted, it is substituted with at least one substituent group. Inembodiments, when B is substituted, it is substituted with at least onesize-limited substituent group. In embodiments, when B is substituted,it is substituted with at least one lower substituent group.

In embodiments, B is a divalent cytosine or a derivative thereof,divalent guanine or a derivative thereof, divalent adenine or aderivative thereof, divalent thymine or a derivative thereof, divalenturacil or a derivative thereof, divalent hypoxanthine or a derivativethereof, divalent xanthine or a derivative thereof, divalent7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or aderivative thereof, divalent 5-methylcytosine or a derivative thereof,or divalent 5-hydroxymethylcytosine or a derivative thereof. Inembodiments, B is a divalent cytosine or a derivative thereof. Inembodiments, B is a divalent guanine or a derivative thereof. Inembodiments, B is a divalent adenine or a derivative thereof. Inembodiments, B is a divalent thymine or a derivative thereof. Inembodiments, B is a divalent uracil or a derivative thereof. Inembodiments, B is a divalent hypoxanthine or a derivative thereof. Inembodiments, B is a divalent xanthine or a derivative thereof. Inembodiments, B is a divalent 7-methylguanine or a derivative thereof. Inembodiments, B is a divalent 5,6-dihydrouracil or a derivative thereof.In embodiments, B is a divalent 5-methylcytosine or a derivativethereof. In embodiments, B is a divalent 5-hydroxymethylcytosine or aderivative thereof. In embodiments, B is a divalent cytosine. Inembodiments, B is a divalent guanine. In embodiments, B is a divalentadenine. In embodiments, B is a divalent thymine. In embodiments, B is adivalent uracil. In embodiments, B is a divalent hypoxanthine. Inembodiments, B is a divalent xanthine. In embodiments, B is a divalent7-methylguanine. In embodiments, B is a divalent 5,6-dihydrouracil. Inembodiments, B is a divalent 5-methylcytosine. In embodiments, B is adivalent 5-hydroxymethylcytosine.

In embodiments, the compound is

wherein, L¹⁰⁰ is a cleavable linker including

B is a divalent cytosine, divalent guanine, divalent adenine, divalentthymine, divalent uracil, divalent hypoxanthine, divalent xanthine,divalent 7-methylguanine, divalent 5,6-dihydrouracil, divalent5-methylcytosine, or divalent 5-hydroxymethylcytosine or a derivativethereof and R³ and R⁴ are as defined herein. In embodiments, thecompound is

wherein, L¹⁰⁰ is a cleavable linker including

and R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ are as defined herein.

In embodiments, R⁵ is hydrogen, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, substituted or unsubstituted alkyl(e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), substituted orunsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), substituted or unsubstituted heterocycloalkyl(e.g., 3 to 8, 3 to 6, or 5 to 6 membered), substituted or unsubstitutedaryl (e.g., C₆-C₁₀, C₁₀, or phenyl), or substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments,R⁵ is H. In embodiments, R⁵ is substituted or unsubstituted alkyl (e.g.,C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄). In embodiments, R⁵ issubstituted or unsubstituted alkenyl (e.g., C₂-C₂₀, C₁₀-C₂₀, C₂-C₈,C₂-C₆, or C₂-C₄). In embodiments, R⁵ is substituted or unsubstitutedalkynyl (e.g., C₂-C₂₀, C₁₀-C₂₀, C₂-C₈, C₂-C₆, or C₂-C₄). In embodiments,R⁵ is substituted or unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20,2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), substituted orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), substituted orunsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6membered), substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or substituted or unsubstituted heteroaryl (e.g., 5 to 10, 5 to9, or 5 to 6 membered). In embodiments, R⁵ is hydrogen, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,R^(5A)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈,C₁-C₆, or C₁-C₄), R^(SA)-substituted or unsubstituted heteroalkyl (e.g.,2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),R^(SA)-substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), R^(SA)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to8, 3 to 6, or 5 to 6 membered), R^(5A)-substituted or unsubstituted aryl(e.g., C₆-C₁₀, C₁₀, or phenyl), or R^(SA)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

R^(5A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, R^(5B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(5B)-substituted or unsubstituted heteroalkyl(e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),R^(5B)-substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), R^(5B)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to8, 3 to 6, or 5 to 6 membered), R^(5B)-substituted or unsubstituted aryl(e.g., C₆-C₁₀, C₁₀, or phenyl), or R^(5B)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

R^(5B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₅Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁺, —OPO₃H⁻, —SCN,—ONO₂, R^(5C)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(5C)-substituted or unsubstituted heteroalkyl(e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),R^(5C)-substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), R^(5C)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to8, 3 to 6, or 5 to 6 membered), R^(5C)-substituted or unsubstituted aryl(e.g., C₆-C₁₀, C₁₀, or phenyl), or R^(5C)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

R^(5C) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₅C₁, —CH₅Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₅C₁,—OCH₅Br, —OCH₂T, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, orC₁-C₄), unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to8, 2 to 6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈,C₃-C₆, or C₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6,or 5 to 6 membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl),or unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, R⁵ is hydrogen. In embodiments, R⁵ is —CH₃. Inembodiments, R⁵ is unsubstituted C₁-C₆ alkyl. In embodiments, R⁵ is —For —Cl. In embodiments, R⁵ is a halogen. In embodiments, R⁵ is —CN. Inembodiments, R⁵ is phenyl.

In embodiments, R⁵ is substituted or unsubstituted C₁-C₆ alkyl. Inembodiments, R⁵ is substituted or unsubstituted C₁-C₆ alkyl. Inembodiments, R⁵ is substituted or unsubstituted C₁ alkyl. Inembodiments, R⁵ is substituted or unsubstituted C₂ alkyl. Inembodiments, R⁵ is substituted or unsubstituted C₃ alkyl. Inembodiments, R⁵ is substituted or unsubstituted C₄ alkyl. Inembodiments, R⁵ is substituted or unsubstituted C₅ alkyl. Inembodiments, R⁵ is substituted or unsubstituted C₆ falkyl. Inembodiments, R⁵ is substituted C₁ alkyl. In embodiments, R⁵ issubstituted C₂ alkyl. In embodiments, R⁵ is substituted C₃ alkyl. Inembodiments, R⁵ is substituted C₄ alkyl. In embodiments, R⁵ issubstituted C₅ alkyl. In embodiments, R⁵ is substituted C₆ alkyl. Inembodiments, R⁵ is unsubstituted C₁ alkyl. In embodiments, R⁵ isunsubstituted C₂ alkyl. In embodiments, R⁵ is unsubstituted C₃ alkyl. Inembodiments, R⁵ is unsubstituted C₄ alkyl. In embodiments, R⁵ isunsubstituted C₅ alkyl. In embodiments, R⁵ is unsubstituted C₆ alkyl.

In embodiments, R⁵ is substituted or unsubstituted 2 to 8 memberedheteroalkyl. In embodiments, R⁵ is substituted or unsubstituted 2 to 6membered heteroalkyl. In embodiments, R⁵ is substituted or unsubstituted2 to 4 membered heteroalkyl. In embodiments, R⁵ is substituted 2 to 8membered heteroalkyl. In embodiments, R⁵ is substituted 2 to 6 memberedheteroalkyl. In embodiments, R⁵ is substituted 2 to 4 memberedheteroalkyl. In embodiments, R⁵ is unsubstituted 2 to 8 memberedheteroalkyl. In embodiments, R⁵ is unsubstituted 2 to 6 memberedheteroalkyl. In embodiments, R⁵ is unsubstituted 2 to 4 memberedheteroalkyl.

In embodiments, R⁵ is substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered), substituted orunsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or substituted orunsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5to 6 membered).

In embodiments, R⁵ is substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). In embodiments, R⁵ is substitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). In embodiments, R⁵ isan unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). Inembodiments, R⁵ is substituted or unsubstituted heterocycloalkyl (e.g.,3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered). In embodiments, R⁵ is substituted heterocycloalkyl(e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5membered, or 5 to 6 membered). In embodiments, R⁵ is an unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered).

In embodiments, R⁵ is substituted or unsubstituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁵ is substituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁵ is unsubstituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁵ is unsubstituted phenyl. Inembodiments, R⁵ is substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered). In embodiments, R⁵ is substitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments,R⁵ is unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6membered). In embodiments, R⁵ is a substituted or unsubstituted 5membered heteroaryl. In embodiments, R⁵ is a substituted orunsubstituted 6 membered heteroaryl. In embodiments, R⁵ is a substitutedor unsubstituted 5 membered heteroaryl. In embodiments, R⁵ is anunsubstituted 5 membered heteroaryl. In embodiments, R⁵ is anunsubstituted 6 membered heteroaryl. In embodiments, R⁵ is anunsubstituted 7 membered heteroaryl.

In embodiments, R⁶ is hydrogen, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, substituted or unsubstituted alkyl(e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), substituted orunsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), substituted or unsubstituted heterocycloalkyl(e.g., 3 to 8, 3 to 6, or 5 to 6 membered), substituted or unsubstitutedaryl (e.g., C₆-C₁₀, C₁₀, or phenyl), or substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments,R⁶ is H. In embodiments, R⁶ is substituted or unsubstituted alkyl (e.g.,C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄). In embodiments, R⁶ issubstituted or unsubstituted alkenyl (e.g., C₂-C₂₀, C₁₀-C₂₀, C₂-C₈,C₂-C₆, or C₂-C₄). In embodiments, R⁶ is substituted or unsubstitutedalkynyl (e.g., C₂-C₂₀, C₁₀-C₂₀, C₂-C₈, C₂-C₆, or C₂-C₄). In embodiments,R⁶ is substituted or unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20,2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), substituted orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), substituted orunsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6membered), substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or substituted or unsubstituted heteroaryl (e.g., 5 to 10, 5 to9, or 5 to 6 membered). In embodiments, R⁶ is hydrogen, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,R^(6A)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈,C₁-C₆, or C₁-C₄), R^(6A)-substituted or unsubstituted heteroalkyl (e.g.,2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),R^(6A)-substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), R^(6A)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to8, 3 to 6, or 5 to 6 membered), R^(6A)-substituted or unsubstituted aryl(e.g., C₆-C₁₀, C₁₀, or phenyl), or R^(6A)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

R^(6A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, R^(6B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(6B)-substituted or unsubstituted heteroalkyl(e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),R^(6B)-substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), R^(6B)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to8, 3 to 6, or 5 to 6 membered), R^(6B)-substituted or unsubstituted aryl(e.g., C₆-C₁₀, C₁₀, or phenyl), or R^(6B)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

R^(6B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH_(5C)l,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H″, —SCN,—ONO₂, R^(6C)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(6C)-substituted or unsubstituted heteroalkyl(e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),R^(6C)-substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), R^(6C)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to8, 3 to 6, or 5 to 6 membered), R^(6C)-substituted or unsubstituted aryl(e.g., C₆-C₁₀, C₁₀, or phenyl), or R^(6C)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

R^(6C) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH_(5C)l, —CH₅Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH_(5C)l,—OCH_(5B)r, —OCH₂I, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, orC₁-C₄), unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to8, 2 to 6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈,C₃-C₆, or C₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6,or 5 to 6 membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl),or unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, R⁶ is hydrogen. In embodiments, R⁶ is —CH₃. Inembodiments, R⁶ is unsubstituted C₁-C₆ alkyl. In embodiments, R⁶ is —For —Cl. In embodiments, R⁶ is a halogen. In embodiments, R⁶ is —CN. Inembodiments, R⁶ is phenyl.

In embodiments, R⁶ is substituted or unsubstituted C₁-C₆ alkyl. Inembodiments, R⁶ is substituted or unsubstituted C₁-C₆ alkyl. Inembodiments, R⁶ is substituted or unsubstituted C₁ alkyl. Inembodiments, R⁶ is substituted or unsubstituted C₂ alkyl. Inembodiments, R⁶ is substituted or unsubstituted C₃ alkyl. Inembodiments, R⁶ is substituted or unsubstituted C₄ alkyl. Inembodiments, R⁶ is substituted or unsubstituted C₅ alkyl. Inembodiments, R⁶ is substituted or unsubstituted C₆ alkyl. Inembodiments, R⁶ is substituted C₁ alkyl. In embodiments, R⁶ issubstituted C₂ alkyl. In embodiments, R⁶ is substituted C₃ alkyl. Inembodiments, R⁶ is substituted C₄ alkyl. In embodiments, R⁶ issubstituted C₅ alkyl. In embodiments, R⁶ is substituted C₆ alkyl. Inembodiments, R⁶ is unsubstituted C₁ alkyl. In embodiments, R⁶ isunsubstituted C₂ alkyl. In embodiments, R⁶ is unsubstituted C₃ alkyl. Inembodiments, R⁶ is unsubstituted C₄ alkyl. In embodiments, R⁶ isunsubstituted C₅ alkyl. In embodiments, R⁶ is unsubstituted C₆ alkyl.

In embodiments, R⁶ is substituted or unsubstituted 2 to 8 memberedheteroalkyl. In embodiments, R⁶ is substituted or unsubstituted 2 to 6membered heteroalkyl. In embodiments, R⁶ is substituted or unsubstituted2 to 4 membered heteroalkyl. In embodiments, R⁶ is substituted 2 to 8membered heteroalkyl. In embodiments, R⁶ is substituted 2 to 6 memberedheteroalkyl. In embodiments, R⁶ is substituted 2 to 4 memberedheteroalkyl. In embodiments, R⁶ is unsubstituted 2 to 8 memberedheteroalkyl. In embodiments, R⁶ is unsubstituted 2 to 6 memberedheteroalkyl. In embodiments, R⁶ is unsubstituted 2 to 4 memberedheteroalkyl.

In embodiments, R⁶ is substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered), substituted orunsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or substituted orunsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5to 6 membered).

In embodiments, R⁶ is substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). In embodiments, R⁶ is substitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). In embodiments, R⁶ isan unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). Inembodiments, R⁶ is substituted or unsubstituted heterocycloalkyl (e.g.,3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered). In embodiments, R⁶ is substituted heterocycloalkyl(e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5membered, or 5 to 6 membered). In embodiments, R⁶ is an unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered).

In embodiments, R⁶ is substituted or unsubstituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁶ is substituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁶ is unsubstituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁶ is unsubstituted phenyl. Inembodiments, R⁶ is substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered). In embodiments, R⁶ is substitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments,R⁶ is unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6membered). In embodiments, R⁶ is a substituted or unsubstituted 5membered heteroaryl. In embodiments, R⁶ is a substituted orunsubstituted 6 membered heteroaryl. In embodiments, R⁶ is a substitutedor unsubstituted 5 membered heteroaryl. In embodiments, R⁶ is anunsubstituted 5 membered heteroaryl. In embodiments, R⁶ is anunsubstituted 6 membered heteroaryl. In embodiments, R⁶ is anunsubstituted 7 membered heteroaryl.

In embodiments, R⁵ and R⁶ are both hydrogen. In embodiments, R⁵ ishydrogen and R⁶ is —CH₃. In embodiments, R⁵ is hydrogen and R⁶ isunsubstituted C₁-C₆ alkyl. In embodiments, R⁵ is hydrogen and R⁶ is —F.In embodiments, R⁵ is hydrogen and R⁶ is —Cl. In embodiments, R⁵ ishydrogen and R⁶ is a halogen. In embodiments, R⁵ is hydrogen and R⁶ is—CN. In embodiments, R⁵ is hydrogen and R⁶ is phenyl. In embodiments,both R⁵ and R⁶ are —CH₃. In embodiments, R⁵ is unsubstituted C₁-C₆ alkyland R⁶ is unsubstituted C₁-C₆ alkyl. In embodiments, R⁵ is CH₃ and R⁶ is—F. In embodiments, R⁵ is CH₃ and R⁶ is —Cl. In embodiments, R⁵ is CH₃and R⁶ is a halogen. In embodiments, R⁵ is CH₃ and R⁶ is —CN. Inembodiments, R⁵ is CH₃ and R⁶ is phenyl.

In embodiments, R⁷ is hydrogen. In embodiments, R⁷ is substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.

In embodiments, R⁷ is hydrogen. In embodiments, R⁷ is C₁-C₆ alkyl. Inembodiments, R⁷ is C₁ alkyl. In embodiments, R⁷ is C₂ alkyl. Inembodiments, R⁷ is C₃ alkyl. In embodiments, R⁷ is C₄ alkyl. Inembodiments, R⁷ is C₅ alkyl. In embodiments, R⁷ is C₆ alkyl. Inembodiments, R⁷ is phenyl. In embodiments, R⁷ is substituted phenyl.

In embodiments, R⁷ is hydrogen, substituted or unsubstituted alkyl(e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄) substituted orunsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered), substituted orunsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), or substituted orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, R⁷ is unsubstituted C₁-C₆ or unsubstituted C₁-C₄ alkyl.In embodiments, R⁷ is unsubstituted C₁-C₄ alkyl. In embodiments, R⁷ isunsubstituted C₁-C₆ alkyl. In embodiments, R⁷ is unsubstituted methyl.In embodiments, R⁷ is unsubstituted C₂ alkyl. In embodiments, R⁷ isunsubstituted C₃ alkyl. In embodiments, R⁷ is unsubstituted C₄ alkyl. Inembodiments, R⁷ is unsubstituted C₅ alkyl. In embodiments, R⁷ isunsubstituted C₆ alkyl.

In embodiments, R⁷ is substituted or unsubstituted heteroalkyl (e.g., 3to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered). In embodiments, R⁷ is substituted heteroalkyl (e.g., 3to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered). In embodiments, R⁷ is an unsubstituted heteroalkyl(e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5membered, or 5 to 6 membered).

In embodiments, R⁷ is substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered), substituted orunsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or substituted orunsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5to 6 membered).

In embodiments, R⁷ is substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). In embodiments, R⁷ is substitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). In embodiments, R⁷ isan unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). Inembodiments, R⁷ is substituted or unsubstituted heterocycloalkyl (e.g.,3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered). In embodiments, R⁷ is substituted heterocycloalkyl(e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5membered, or 5 to 6 membered). In embodiments, R⁷ is an unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered).

In embodiments, R⁷ is unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆,C₄-C₆, or C₅-C₆). In embodiments, R⁷ is unsubstituted C₃-C₈ cycloalkyl.In embodiments, R⁷ is unsubstituted C₃-C₆ cycloalkyl. In embodiments, R⁷is unsubstituted C₄-C₆ cycloalkyl. In embodiments, R⁷ is unsubstitutedC₅-C₆ cycloalkyl. In embodiments, R⁷ is unsubstituted cyclopropyl. Inembodiments, R⁷ is unsubstituted cyclobutyl. In embodiments, R⁷ isunsubstituted cyclopentyl. In embodiments, R⁷ is unsubstitutedcyclohexyl. In embodiments, R⁷ is unsubstituted cycloheptyl. Inembodiments, R⁷ is unsubstituted cyclooctyl.

In embodiments, R⁷ is substituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆,or C₅-C₆). In embodiments, R⁷ is substituted C₃-C₈ cycloalkyl. Inembodiments, R⁷ is substituted C₃-C₆ cycloalkyl. In embodiments, R⁷ issubstituted C₄-C₆ cycloalkyl. In embodiments, R⁷ is substituted C₅-C₆cycloalkyl. In embodiments, R⁷ is substituted cyclopropyl. Inembodiments, R⁷ is substituted cyclobutyl. In embodiments, R⁷ issubstituted cyclopentyl. In embodiments, R⁷ is substituted cyclohexyl.In embodiments, R⁷ is substituted cycloheptyl. In embodiments, R⁷ issubstituted cyclooctyl.

In embodiments, R⁷ is substituted or unsubstituted heterocycloalkyl(e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5membered, or 5 to 6 membered). In embodiments, R⁷ is substitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered). In embodiments, R⁷ is anunsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered,4 to 6 membered, 4 to 5 membered, or 5 to 6 membered).

In embodiments, R⁷ is unsubstituted 3 to 8 membered heterocycloalkyl. Inembodiments, R⁷ is unsubstituted 3 to 6 membered heterocycloalkyl. Inembodiments, R⁷ is unsubstituted 4 to 6 membered heterocycloalkyl. Inembodiments, R⁷ is unsubstituted 4 to 5 membered heterocycloalkyl. Inembodiments, R⁷ is unsubstituted 5 to 6 membered heterocycloalkyl. Inembodiments, R⁷ is a substituted 3 to 8 membered heterocycloalkyl. Inembodiments, R⁷ is a substituted 3 to 6 membered heterocycloalkyl. Inembodiments, R⁷ is a substituted 4 to 6 membered heterocycloalkyl. Inembodiments, R⁷ is a substituted 4 to 5 membered heterocycloalkyl. Inembodiments, R⁷ is a substituted 5 to 6 membered heterocycloalkyl.

In embodiments, R⁷ is substituted or unsubstituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁷ is substituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁷ is unsubstituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁷ is unsubstituted phenyl. Inembodiments, R⁷ is substituted phenyl. In embodiments, R⁷ isunsubstituted or substituted naphthyl. In embodiments, R⁷ isunsubstituted naphthyl. In embodiments, R⁷ is substituted naphthyl. Inembodiments, R⁷ is 1-naphthyl, 2-naphthyl or 4-biphenyl.

In embodiments, R⁷ is substituted or unsubstituted heteroaryl (e.g., 5to 10, 5 to 9, or 5 to 6 membered). In embodiments, R⁷ is substitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments,R⁷ is unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6membered). In embodiments, R⁷ is a substituted or unsubstituted 5membered heteroaryl. In embodiments, R⁷ is a substituted orunsubstituted 6 membered heteroaryl. In embodiments, R⁷ is a substitutedor unsubstituted 7 membered heteroaryl. In embodiments, R⁷ is anunsubstituted 5 membered heteroaryl. In embodiments, R⁷ is a pyrrolyl,pyrazolyl, triazinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl,furyl, thienyl or pyridyl. In embodiments, R⁷ is a 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl or 4-imidazolyl. Inembodiments, R⁷ is a 2-oxazolyl, 4-oxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl or5-isoxazolyl. In embodiments, R⁷ is an unsubstituted 6 memberedheteroaryl. In embodiments, R⁷ is a pyrimidyl, pyridazinyl, pyrimidinylor pyrazinyl. In embodiments, R⁷ is 2-pyrimidyl, 3-pyrimidyl or4-pyrimidyl. In embodiments, R⁷ is an unsubstituted 7 memberedheteroaryl. In embodiments, R⁷ is purinyl, benzothiazolyl, benzoxazoylbenzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl,benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl,2-phenyl-4-oxazolyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl,5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl or 6-quinolyl.

In embodiments, R⁷ is hydrogen, R^(7A)-substituted or unsubstitutedalkyl, R^(7A)-substituted or unsubstituted heteroalkyl,R^(7A)-substituted or unsubstituted cycloalkyl, R^(7A)-substituted orunsubstituted heterocycloalkyl, R^(7A)-substituted or unsubstitutedaryl, R^(7A)-substituted or unsubstituted heteroaryl.

R^(7A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —OPO₃H, R^(7B)-substituted orunsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),R^(7B)-substituted or unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20,2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), R^(7B)-substituted orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), 10³-substitutedor unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6membered), R^(7B)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀,or phenyl), or R^(7B)-substituted or unsubstituted heteroaryl (e.g., 5to 10, 5 to 9, or 5 to 6 membered). In embodiments, R^(7A) isindependently halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂,—CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,—OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I,—OCH₂F, —N₃, —SF₅, or —OPO₃H.

R^(7B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, unsubstituted alkyl (e.g., C₁-C₂₀,C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), unsubstituted heteroalkyl (e.g., 2 to20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), unsubstitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, R⁸ is hydrogen. In embodiments, R⁸ is substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.

In embodiments, R⁸ is hydrogen. In embodiments, R⁸ is C₁-C₆ alkyl. Inembodiments, R⁸ is C₁ alkyl. In embodiments, R⁸ is C₂ alkyl. Inembodiments, R⁸ is C₃ alkyl. In embodiments, R⁸ is C₄ alkyl. Inembodiments, R⁸ is C₅ alkyl. In embodiments, R⁸ is C₆ alkyl.

In embodiments, R⁸ is phenyl. In embodiments, R⁸ is substituted phenyl.

In embodiments, R⁸ is hydrogen, substituted or unsubstituted alkyl(e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), substituted orunsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered), substituted orunsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), substituted orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, R⁸ is unsubstituted C₁-C₆ or unsubstituted C₁-C₄ alkyl.In embodiments, R⁸ is unsubstituted C₁-C₄ alkyl. In embodiments, R⁸ isunsubstituted C₁-C₆ alkyl. In embodiments, R⁸ is unsubstituted methyl.In embodiments, R⁸ is unsubstituted C₂ alkyl. In embodiments, R⁸ isunsubstituted C₃ alkyl. In embodiments, R⁸ is unsubstituted C₄ alkyl. Inembodiments, R⁸ is unsubstituted C₅ alkyl. In embodiments, R8 isunsubstituted C₆ alkyl.

In embodiments, R⁸ is substituted or unsubstituted heteroalkyl (e.g., 3to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered). In embodiments, R⁸ is substituted heteroalkyl (e.g., 3to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered). In embodiments, R⁸ is an unsubstituted heteroalkyl(e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5membered, or 5 to 6 membered).

In embodiments, R⁸ is substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered), substituted orunsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or substituted orunsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5to 6 membered).

In embodiments, R⁸ is substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). In embodiments, R⁸ is substitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). In embodiments, R⁸ isan unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). Inembodiments, R⁸ is substituted or unsubstituted heterocycloalkyl (e.g.,3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered). In embodiments, R⁸ is substituted heterocycloalkyl(e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5membered, or 5 to 6 membered). In embodiments, R⁸ is an unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered).

In embodiments, R⁸ is unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆,C₄-C₆, or C₅-C₆). In embodiments, R⁸ is unsubstituted C₃-C₈ cycloalkyl.In embodiments, R⁸ is unsubstituted C₃-C₆ cycloalkyl. In embodiments, R⁸is unsubstituted C₄-C₆ cycloalkyl. In embodiments, R⁸ is unsubstitutedC₅-C₆ cycloalkyl. In embodiments, R⁸ is unsubstituted cyclopropyl. Inembodiments, R⁸ is unsubstituted cyclobutyl. In embodiments, R⁸ isunsubstituted cyclopentyl. In embodiments, R⁸ is unsubstitutedcyclohexyl. In embodiments, R⁸ is unsubstituted cycloheptyl. Inembodiments, R⁸ is unsubstituted cyclooctyl.

In embodiments, R⁸ is substituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆,or C₅-C₆). In embodiments, R⁸ is substituted C₃-C₈ cycloalkyl. Inembodiments, R⁸ is substituted C₃-C₆ cycloalkyl. In embodiments, R⁸ issubstituted C₄-C₆ cycloalkyl. In embodiments, R⁸ is substituted C₅-C₆cycloalkyl. In embodiments, R⁸ is substituted cyclopropyl. Inembodiments, R⁸ is substituted cyclobutyl. In embodiments, R⁸ issubstituted cyclopentyl. In embodiments, R⁸ is substituted cyclohexyl.In embodiments, R⁸ is substituted cycloheptyl. In embodiments, R⁸ issubstituted cyclooctyl.

In embodiments, R⁸ is substituted or unsubstituted heterocycloalkyl(e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5membered, or 5 to 6 membered). In embodiments, R⁸ is substitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered). In embodiments, R⁸ is anunsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered,4 to 6 membered, 4 to 5 membered, or 5 to 6 membered).

In embodiments, R⁸ is unsubstituted 3 to 8 membered heterocycloalkyl. Inembodiments, R⁸ is unsubstituted 3 to 6 membered heterocycloalkyl. Inembodiments, R⁸ is unsubstituted 4 to 6 membered heterocycloalkyl. Inembodiments, R⁸ is unsubstituted 4 to 5 membered heterocycloalkyl. Inembodiments, R⁸ is unsubstituted 5 to 6 membered heterocycloalkyl. Inembodiments, R⁸ is a substituted 3 to 8 membered heterocycloalkyl. Inembodiments, R⁸ is a substituted 3 to 6 membered heterocycloalkyl. Inembodiments, R⁸ is a substituted 4 to 6 membered heterocycloalkyl. Inembodiments, R⁸ is a substituted 4 to 5 membered heterocycloalkyl. Inembodiments, R⁸ is a substituted 5 to 6 membered heterocycloalkyl.

In embodiments, R⁸ is substituted or unsubstituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁸ is substituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁸ is unsubstituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁸ is unsubstituted phenyl. Inembodiments, R⁸ is substituted phenyl. In embodiments, R⁸ isunsubstituted or substituted naphthyl. In embodiments, R⁸ isunsubstituted naphthyl. In embodiments, R⁸ is substituted naphthyl. Inembodiments, R⁸ is 1-naphthyl, 2-naphthyl or 4-biphenyl.

In embodiments, R⁸ is substituted or unsubstituted heteroaryl (e.g., 5to 10, 5 to 9, or 5 to 6 membered). In embodiments, R⁸ is substitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments,R⁸ is unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6membered). In embodiments, R⁸ is a substituted or unsubstituted 5membered heteroaryl. In embodiments, R⁸ is a substituted orunsubstituted 6 membered heteroaryl. In embodiments, R⁸ is a substitutedor unsubstituted 7 membered heteroaryl. In embodiments, R⁸ is anunsubstituted 5 membered heteroaryl. In embodiments, R⁸ is a pyrrolyl,pyrazolyl, triazinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl,furyl, thienyl or pyridyl. In embodiments, R⁸ is a 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl or 4-imidazolyl. Inembodiments, R⁸ is a 2-oxazolyl, 4-oxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl or5-isoxazolyl. In embodiments, R⁸ is an unsubstituted 6 memberedheteroaryl. In embodiments, R⁸ is a pyrimidyl, pyridazinyl, pyrimidinylor pyrazinyl. In embodiments, R⁸ is 2-pyrimidyl, 3-pyrimidyl or4-pyrimidyl. In embodiments, R⁸ is an unsubstituted 7 memberedheteroaryl. In embodiments, R⁸ is purinyl, benzothiazolyl, benzoxazoylbenzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl,benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl,2-phenyl-4-oxazolyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl,5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl or 6-quinolyl.

In embodiments, R⁸ is hydrogen, R^(8A)-substituted or unsubstitutedalkyl, R^(8A)-substituted or unsubstituted heteroalkyl,R^(8A)-substituted or unsubstituted cycloalkyl, R^(8A)-substituted orunsubstituted heterocycloalkyl, R^(8A)-substituted or unsubstitutedaryl, R^(8A)-substituted or unsubstituted heteroaryl.

R^(8A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —OPO₃H, R^(8B)-substituted orunsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),R^(8B)-substituted or unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20,2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), R^(8B)-substituted orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆),R^(8B)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to6, or 5 to 6 membered), R^(8B)-substituted or unsubstituted aryl (e.g.,C₆-C₁₀, C₁₀, or phenyl), or R^(8B)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments,R^(8A) is independently halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, or —OPO₃H.

R^(8B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, unsubstituted alkyl (e.g., C₁-C₂₀,C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), unsubstituted heteroalkyl (e.g., 2 to20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), unsubstitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, R⁹ is hydrogen. In embodiments, R⁹ is substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.

In embodiments, R⁹ is hydrogen. In embodiments, R⁹ is C₁-C₆ alkyl. Inembodiments, R⁹ is C₁ alkyl. In embodiments, R⁹ is C₂ alkyl. Inembodiments, R⁹ is C₃ alkyl. In embodiments, R⁹ is C₄ alkyl. Inembodiments, R⁹ is C₅ alkyl. In embodiments, R⁹ is C₆ alkyl. Inembodiments, R⁹ is phenyl. In embodiments, R⁹ is substituted phenyl.

In embodiments, R⁹ is hydrogen, substituted or unsubstituted alkyl(e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), substituted orunsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered), substituted orunsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), substituted orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, R⁹ is unsubstituted C₁-C₆ or unsubstituted C₁-C₄ alkyl.In embodiments, R⁹ is unsubstituted C₁-C₄ alkyl. In embodiments, R⁹ isunsubstituted C₁-C₆ alkyl. In embodiments, R⁹ is unsubstituted methyl.In embodiments, R⁹ is unsubstituted C₂ alkyl. In embodiments, R⁹ isunsubstituted C₃ alkyl. In embodiments, R⁹ is unsubstituted C₄ alkyl. Inembodiments, R⁹ is unsubstituted C₅ alkyl. In embodiments, R⁹ isunsubstituted C₆ alkyl.

In embodiments, R⁹ is substituted or unsubstituted heteroalkyl (e.g., 3to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered). In embodiments, R⁹ is substituted heteroalkyl (e.g., 3to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered). In embodiments, R⁹ is an unsubstituted heteroalkyl(e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5membered, or 5 to 6 membered).

In embodiments, R⁹ is substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered), substituted orunsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or substituted orunsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5to 6 membered).

In embodiments, R⁹ is substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). In embodiments, R⁹ is substitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). In embodiments, R⁹ isan unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). Inembodiments, R⁹ is substituted or unsubstituted heterocycloalkyl (e.g.,3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered). In embodiments, R⁹ is substituted heterocycloalkyl(e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5membered, or 5 to 6 membered). In embodiments, R⁹ is an unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered).

In embodiments, R⁹ is unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆,C₄-C₆, or C₅-C₆). In embodiments, R⁹ is unsubstituted C₃-C₈ cycloalkyl.In embodiments, R⁹ is unsubstituted C₃-C₆ cycloalkyl. In embodiments, R⁹is unsubstituted C₄-C₆ cycloalkyl. In embodiments, R⁹ is unsubstitutedC₅-C₆ cycloalkyl. In embodiments, R⁹ is unsubstituted cyclopropyl. Inembodiments, R⁹ is unsubstituted cyclobutyl. In embodiments, R⁹ isunsubstituted cyclopentyl. In embodiments, R⁹ is unsubstitutedcyclohexyl. In embodiments, R⁹ is unsubstituted cycloheptyl. Inembodiments, R⁹ is unsubstituted cyclooctyl.

In embodiments, R⁹ is substituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆,or C₅-C₆). In embodiments, R⁹ is substituted C₃-C₈ cycloalkyl. Inembodiments, R⁹ is substituted C₃-C₆ cycloalkyl. In embodiments, R⁹ issubstituted C₄-C₆ cycloalkyl. In embodiments, R⁹ is substituted C₅-C₆cycloalkyl. In embodiments, R⁹ is substituted cyclopropyl. Inembodiments, R⁹ is substituted cyclobutyl. In embodiments, R⁹ issubstituted cyclopentyl. In embodiments, R⁹ is substituted cyclohexyl.In embodiments, R⁹ is substituted cycloheptyl. In embodiments, R⁹ issubstituted cyclooctyl.

In embodiments, R⁹ is substituted or unsubstituted heterocycloalkyl(e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5membered, or 5 to 6 membered). In embodiments, R⁹ is substitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered). In embodiments, R⁹ is anunsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered,4 to 6 membered, 4 to 5 membered, or 5 to 6 membered).

In embodiments, R⁹ is unsubstituted 3 to 8 membered heterocycloalkyl. Inembodiments, R⁹ is unsubstituted 3 to 6 membered heterocycloalkyl. Inembodiments, R⁹ is unsubstituted 4 to 6 membered heterocycloalkyl. Inembodiments, R⁹ is unsubstituted 4 to 5 membered heterocycloalkyl. Inembodiments, R⁹ is unsubstituted 5 to 6 membered heterocycloalkyl. Inembodiments, R⁹ is a substituted 3 to 8 membered heterocycloalkyl. Inembodiments, R⁹ is a substituted 3 to 6 membered heterocycloalkyl. Inembodiments, R⁹ is a substituted 4 to 6 membered heterocycloalkyl. Inembodiments, R⁹ is a substituted 4 to 5 membered heterocycloalkyl. Inembodiments, R⁹ is a substituted 5 to 6 membered heterocycloalkyl.

In embodiments, R⁹ is substituted or unsubstituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁹ is substituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁹ is unsubstituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁹ is unsubstituted phenyl. Inembodiments, R⁹ is substituted phenyl. In embodiments, R⁹ isunsubstituted or substituted naphthyl. In embodiments, R⁹ isunsubstituted naphthyl. In embodiments, R⁹ is substituted naphthyl. Inembodiments, R⁹ is 1-naphthyl, 2-naphthyl or 4-biphenyl.

In embodiments, R⁹ is substituted or unsubstituted heteroaryl (e.g., 5to 10, 5 to 9, or 5 to 6 membered). In embodiments, R⁹ is substitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments,R⁹ is unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6membered). In embodiments, R⁹ is a substituted or unsubstituted 5membered heteroaryl. In embodiments, R⁹ is a substituted orunsubstituted 6 membered heteroaryl. In embodiments, R⁹ is a substitutedor unsubstituted 7 membered heteroaryl. In embodiments, R⁹ is anunsubstituted 5 membered heteroaryl. In embodiments, R⁹ is a pyrrolyl,pyrazolyl, triazinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl,furyl, thienyl or pyridyl. In embodiments, R⁹ is a 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl or 4-imidazolyl. Inembodiments, R⁹ is a 2-oxazolyl, 4-oxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl or5-isoxazolyl. In embodiments, R⁹ is an unsubstituted 6 memberedheteroaryl. In embodiments, R⁹ is a pyrimidyl, pyridazinyl, pyrimidinylor pyrazinyl. In embodiments, R⁹ is 2-pyrimidyl, 3-pyrimidyl or4-pyrimidyl. In embodiments, R⁹ is an unsubstituted 7 memberedheteroaryl. In embodiments, R⁹ is purinyl, benzothiazolyl, benzoxazoylbenzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl,benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl,2-phenyl-4-oxazolyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl,5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl or 6-quinolyl.

In embodiments, R⁹ is hydrogen, R^(9A)-substituted or unsubstitutedalkyl, R^(9A)-substituted or unsubstituted heteroalkyl,R^(9A)-substituted or unsubstituted cycloalkyl, R^(9A)-substituted orunsubstituted heterocycloalkyl, R^(9A)-substituted or unsubstitutedaryl, R^(9A)-substituted or unsubstituted heteroaryl.

R^(9A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —OPO₃H, R^(9B)-substituted orunsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),R^(9B)-substituted or unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20,2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), R^(9B)-substituted orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆),R^(9B)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to6, or 5 to 6 membered), R^(9B)-substituted or unsubstituted aryl (e.g.,C₆-C₁₀, C₁₀, or phenyl), or R^(9B)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments,R^(9A) is independently halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, or —OPO₃H.

R^(9B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, unsubstituted alkyl (e.g., C₁-C₂₀,C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), unsubstituted heteroalkyl (e.g., 2 to20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), unsubstitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, R⁷, R⁸ and R⁹ are all hydrogen. In embodiments, R⁷, R⁸and R⁹ are all —CH₃. In embodiments, R⁷, R⁸ and R⁹ are all C₂ alkyl. Inembodiments, R⁷, R⁸ and R⁹ are all C₃ alkyl. In embodiments, R⁷, R¹ andR⁹ are all C₄ alkyl. In embodiments, R⁷, R⁸ and R⁹ are all C₅ alkyl. Inembodiments, R⁷, R¹ and R⁹ are all C₆ alkyl. In embodiments, one of R⁷,R⁸, and R⁹ is hydrogen and the other two are —CH₃. In embodiments, twoof R⁷, R⁸, and R⁹ are hydrogen and one of R⁷, R⁸, and R⁹ is —CH₃. Inembodiments, one of R⁷, R⁸, and R⁹ is hydrogen and the other two are C₂alkyl. In embodiments, two of R⁷, R⁸, and R⁹ are hydrogen and one of R⁷,R⁸, and R⁹ is C₂ alkyl. In embodiments, one of R⁷, R⁸, and R⁹ ishydrogen and the other two are C₃ alkyl. In embodiments, two of R⁷, R⁸,and R⁹ are hydrogen and one of R⁷, R⁸, and R⁹ is C₃ alkyl. Inembodiments, one of R⁷, R⁸, and R⁹ is hydrogen and the other two are C₄alkyl. In embodiments, two of R⁷, R⁸, and R⁹ are hydrogen and one of R⁷,R⁸, and R⁹ is C₄ alkyl. In embodiments, one of R⁷, R⁸, and R⁹ ishydrogen and the other two are C₅ alkyl. In embodiments, two of R⁷, R⁸,and R⁹ are hydrogen and one of R⁷, R⁸, and R⁹ is C₅ alkyl. Inembodiments, one of R⁷, R⁸, and R⁹ is hydrogen and the other two are C₆alkyl. In embodiments, two of R⁷, R⁸, and R⁹ are hydrogen and one of R⁷,R⁸, and R⁹ is C₆ alkyl. In embodiments, R⁷, R⁸, and R⁹ are independentlyC₁-C₆ alkyl.

In embodiments, R¹⁰ is substituted or unsubstituted alkyl. Inembodiments, R¹⁰ is substituted or unsubstituted C₁-C₈ alkyl. Inembodiments, R¹⁰ is substituted or unsubstituted C₁ alkyl. Inembodiments, R¹⁰ is substituted or unsubstituted C₂ alkyl. Inembodiments, R¹⁰ is substituted or unsubstituted C₃ alkyl. Inembodiments, R¹⁰ is substituted or unsubstituted C₄ alkyl. Inembodiments, R¹⁰ is substituted or unsubstituted C₈ alkyl. Inembodiments, R¹⁰ is substituted or unsubstituted C₆ alkyl. Inembodiments, R¹⁰ is substituted or unsubstituted C₇ alkyl. Inembodiments, R¹⁰ is substituted or unsubstituted C₈ alkyl. Inembodiments, R¹⁰ is substituted C₁ alkyl. In embodiments, R¹⁰ issubstituted C₂ alkyl. In embodiments, R¹⁰ is substituted C₃ alkyl. Inembodiments, R¹⁰ is substituted C₄ alkyl. In embodiments, R¹⁰ issubstituted C₅ alkyl. In embodiments, R¹⁰ is substituted C₆ alkyl. Inembodiments, R¹⁰ is substituted C₇ alkyl. In embodiments, R¹⁰ issubstituted C₈ alkyl. In embodiments, R¹⁰ is unsubstituted C₁ alkyl. Inembodiments, R¹⁰ is unsubstituted C₂ alkyl. In embodiments, R¹⁰ isunsubstituted C₃ alkyl. In embodiments, R¹⁰ is unsubstituted C₄ alkyl.In embodiments, R¹⁰ is unsubstituted C₅ alkyl. In embodiments, R¹⁰ isunsubstituted C₆ alkyl. In embodiments, R¹⁰ is unsubstituted C₇ alkyl.In embodiments, R¹⁰ is unsubstituted C₈ alkyl.

In embodiments, R10 is R^(10A)-substituted or unsubstituted alkyl.

R^(10A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —OPO₃H, R^(10B)-substituted orunsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),R^(10B)-substituted or unsubstituted heteroalkyl (e.g., 2 to 20, 8 to20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), R^(10B)-substituted orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆),R^(10B)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8, 3to 6, or 5 to 6 membered), R^(10B)-substituted or unsubstituted aryl(e.g., C₆-C₁₀, C₁₀, or phenyl), or R^(10B)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments,R^(10A) is independently halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, or —OPO₃H.

R^(10B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, unsubstituted alkyl (e.g., C₁-C₂₀,C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), unsubstituted heteroalkyl (e.g., 2 to20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), unsubstitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, L¹⁰⁰ is a divalent linker. In embodiments, L¹⁰⁰ is adivalent linker including

In embodiments, L¹⁰⁰ is a divalent linker including

In embodiments, L¹⁰⁰ is L¹⁰¹-L¹⁰²-L¹⁰³-L¹⁰⁴-L¹⁰⁵-. In embodiments, L¹⁰¹,L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are independently a bond, —NH—, —S—, —O—,—C(O)—, —C(O)O—, —OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—,—C(S)—, —N═N—, —SS—, substituted or unsubstituted alkylene (e.g.,—CH(OH)— or —C(CH₂)—), substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene, a bioconjugate linker, acleavable linker, a self-immolative linker, a linker capable ofdendritic amplification of signal (e.g., capable of increasingfluorescence by releasing fluorophores from the remainder of thelinker), a trivalent linker, or a self-immolative dendrimer linker(e.g., capable of increasing fluorescence by releasing fluorophores fromthe remainder of the linker). In embodiments, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵are independently a bond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—,—NHC(O)NH—, —C(O)O—, —OC(O)—, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. In embodiments, L¹⁰¹, L¹⁰²,L¹⁰³, L¹⁰⁴, and L¹⁰⁵ independently includes PEG. In embodiments, L¹⁰¹,L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ independently includes

wherein z100 is an integer from 1 to 8. In embodiments, z100 is 1. Inembodiments, z100 is 2. In embodiments, z100 is 3. In embodiments, z100is 4. In embodiments, z100 is 5. In embodiments, z100 is 6. Inembodiments, z100 is 7. In embodiments, z100 is 8. In embodiments, z100is 2 to 8. In embodiments, z100 is 4 to 6.

In embodiments, L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are independently abond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—,—OC(O)—, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene. In embodiments, L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and/or L¹⁰⁵ areindependently a bond, —NH—, —S—, —O—, —C(O)—, —C(O)O—, —OC(O)—,—CH(OH)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—, or—C(CH₂)—.

In embodiments, L¹⁰⁰ is

wherein L¹⁰¹, L¹⁰³, L¹⁰⁴, L¹⁰⁵, R⁵, R⁶, R⁷, and R⁹ are as describedherein. In embodiments, L¹⁰⁰ is

wherein L¹⁰¹, L¹⁰³, L¹⁰⁴, L¹⁰⁵ are as described herein. In embodiments,L¹⁰⁰ is

wherein L¹⁰¹, L¹⁰³, L¹⁰⁴, L¹⁰⁵ are as described herein. In embodiments,L¹⁰⁰ is

wherein L¹⁰¹, L¹⁰³, L¹⁰⁴, L¹⁰⁵ are as described herein.

In embodiments, L¹⁰⁰ is

wherein L¹⁰³, L¹⁰⁴, L¹⁰⁵, R⁵, R⁶, R⁷, R⁸, and R⁹ are as describedherein. In embodiments, L¹⁰⁰ is

wherein L¹⁰³, L¹⁰⁴, L¹⁰⁵ are as described herein. In embodiments, L¹⁰⁰is

wherein L¹⁰³, L¹⁰⁴, L¹⁰⁵ are as described herein. In embodiments, L¹⁰⁰is

wherein L¹⁰³, L¹⁰⁴, L¹⁰⁵ are as described herein.

In embodiments, L¹⁰¹ is a substituted or unsubstituted C₁-C₄ alkylene orsubstituted or unsubstituted 8 to 20 membered heteroalkylene; L¹⁰³ is abond or substituted or unsubstituted 2 to 10 membered heteroalkylene;L¹⁰⁴ is a bond, substituted or unsubstituted 4 to 18 memberedheteroalkylene, or substituted or unsubstituted phenylene; L¹⁰⁵ is abond or substituted or unsubstituted 4 to 18 membered heteroalkylene; R⁵and R⁶ are unsubstituted C₁-C₄ alkyl; and R⁷, R⁸ and R⁹ are substitutedor unsubstituted C₁-C₆ alkyl. In embodiments, L¹⁰¹ is a substituted orunsubstituted C₁-C₄ alkylene or substituted or unsubstituted 8 to 20membered heteroalkylene. In embodiments, L¹⁰³ is a bond or substitutedor unsubstituted 2 to 10 membered heteroalkylene. In embodiments, L¹⁰⁴is a bond, substituted or unsubstituted 4 to 18 membered heteroalkylene,or substituted or unsubstituted phenylene. In embodiments, L¹⁰⁵ is abond or substituted or unsubstituted 4 to 18 membered heteroalkylene.

In embodiments, L¹⁰¹ is a bond, —NH—, —S—, —O—, —C(O)—, —C(O)O—,—OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—,substituted or unsubstituted alkylene (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈,C₁-C₆, or C₁-C₄), substituted or unsubstituted heteroalkylene (e.g., 2to 20 membered, 8 to 20 membered, 2 to 10 membered, 3 to 10 membered, 2to 8 membered, 2 to 6 membered, or 2 to 4 membered), substituted orunsubstituted cycloalkylene (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), substitutedor unsubstituted heterocycloalkylene (e.g., 3 to 8, 3 to 6, or 5 to 6membered), substituted or unsubstituted arylene (e.g., C₆-C₁₀, C₁₀, orphenylene), or substituted or unsubstituted heteroarylene (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

In embodiments, L¹⁰¹ is a bond, —NH—, —S—, —O—, —C(O)—, —C(O)O—,—OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—,R¹⁰¹-substituted or unsubstituted alkylene (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R¹⁰¹-substituted or unsubstitutedheteroalkylene (e.g., 2 to 20 membered, 8 to 20 membered, 2 to 10membered, 3 to 10 membered, 2 to 8 membered, 2 to 6 membered, or 2 to 4membered), R¹⁰¹-substituted or unsubstituted cycloalkylene (e.g., C₃-C₈,C₃-C₆, or C₅-C₆), R¹⁰¹-substituted or unsubstituted heterocycloalkylene(e.g., 3 to 8, 3 to 6, or 5 to 6 membered), R¹⁰¹-substituted orunsubstituted arylene (e.g., C₆-C₁₀, C₁₀, or phenylene), orR¹⁰¹-substituted or unsubstituted heteroarylene (e.g., 5 to 10, 5 to 9,or 5 to 6 membered).

R¹⁰¹ is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(101A)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(101A)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(101A)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(101A)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(101A)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(101A)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

R^(101A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(101B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(101B)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(101B)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(101B)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(101B)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(101B)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

R^(101B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, L¹⁰² is a bond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—,—NHC(O)NH—, —C(O)O—, —OC(O)—, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene.

In embodiments, L¹⁰² is a bond, —NH—, —S—, —O—, —C(O)—, —C(O)O—,—OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—, —SS—,substituted or unsubstituted alkylene (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈,C₁-C₆, or C₁-C₄), substituted or unsubstituted heteroalkylene (e.g., 2to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),substituted or unsubstituted cycloalkylene (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), substituted or unsubstituted heterocycloalkylene (e.g., 3 to 8,3 to 6, or 5 to 6 membered), substituted or unsubstituted arylene (e.g.,C₆-C₁₀, C₁₀, or phenylene), or substituted or unsubstitutedheteroarylene (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). Inembodiments, L¹⁰² is a bond, —NH—, —S—, —O—, —C(O)—, —C(O)O—, —OC(O)—,—NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—, —SS—,R¹⁰²-substituted or unsubstituted alkylene (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R¹⁰²-substituted or unsubstitutedheteroalkylene (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to4 membered), R¹⁰²-substituted or unsubstituted cycloalkylene (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R¹⁰²-substituted or unsubstitutedheterocycloalkylene (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R¹⁰²-substituted or unsubstituted arylene (e.g., C₆-C₁₀, C₁₀, orphenylene), or R¹⁰²-substituted or unsubstituted heteroarylene (e.g., 5to 10, 5 to 9, or 5 to 6 membered). In embodiments, L102 isR¹⁰²-substituted or unsubstituted C₁-C₂₀ alkylene. In embodiments, L102is R¹⁰²-substituted or unsubstituted 2 to 20 membered heteroalkylene. Inembodiments, L102 is R¹⁰²-substituted or unsubstituted C₃-C₈cycloalkylene. In embodiments, L102 is R¹⁰²-substituted or unsubstituted3 to 8 membered heterocycloalkylene. In embodiments, L102 isR¹⁰²-substituted or unsubstituted C₆-C₁₀ arylene. In embodiments, L¹⁰²is R¹⁰²-substituted or unsubstituted phenylene. In embodiments, L¹⁰² isR¹⁰²-substituted or unsubstituted 5 to 10 membered heteroarylene.

R¹⁰² is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(102A)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(102A)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(102A)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(102A)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),10^(02A)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(102A)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

R^(102A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(102B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(102B)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), 10^(02B)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(102B)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(102B)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(102B)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

R^(102B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, L¹⁰³ is a bond, —NH—, —S—, —O—, —C(O)—, —C(O)O—,—OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—, —N═N—,—SS—, R¹⁰³-substituted or unsubstituted alkylene (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R¹⁰³-substituted or unsubstitutedheteroalkylene (e.g., 2 to 20 membered, 8 to 20 membered, 5 to 16membered, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), 10⁰³-substitutedor unsubstituted cycloalkylene (e.g., C₃-C₈, C₃-C₆, or C₅-C₆),R¹⁰³-substituted or unsubstituted heterocycloalkylene (e.g., 3 to 8, 3to 6, or 5 to 6 membered), 10⁰³-substituted or unsubstituted arylene(e.g., C₆-C₁₀, C₁₀, or phenylene), or R¹⁰³-substituted or unsubstitutedheteroarylene (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). Inembodiments, L¹⁰³ is R¹⁰³-substituted or unsubstituted C₁-C₂₀ alkylene.In embodiments, L¹⁰³ is R¹⁰³-substituted or unsubstituted 2 to 20membered heteroalkylene. In embodiments, L¹⁰³ is R¹⁰³-substituted orunsubstituted 5 to 16 membered heteroalkylene. In embodiments, L¹⁰³ isR¹⁰³-substituted or unsubstituted 2 to 10 membered heteroalkylene. Inembodiments, L¹⁰³ is R¹⁰³-substituted or unsubstituted C₃-C₈cycloalkylene. In embodiments, L¹⁰³ is R¹⁰³-substituted or unsubstituted3 to 8 membered heterocycloalkylene. In embodiments, L¹⁰³ isR¹⁰³-substituted or unsubstituted C₆-C₁₀ arylene. In embodiments, L¹⁰³is R¹⁰³-substituted or unsubstituted 5 to 10 membered heteroarylene.

R¹⁰³ is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(103A)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(103A)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(103A)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(103A)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(103A)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(103A)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

R^(103A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(103B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(103B)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(103B)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(103B)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(103B)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(103B)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

R^(103B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, L¹⁰⁴ is a bond, —NH—, —S—, —O—, —C(O)—, —C(O)O—,—OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—,R¹⁰⁴-substituted or unsubstituted alkylene (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R¹⁰⁴-substituted or unsubstitutedheteroalkylene (e.g., 2 to 20 membered, 8 to 20 membered, 5 to 16membered, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), R¹⁰⁴-substitutedor unsubstituted cycloalkylene (e.g., C₃-C₈, C₃-C₆, or C₅-C₆),R₁₀₄-substituted or unsubstituted heterocycloalkylene (e.g., 3 to 8, 3to 6, or 5 to 6 membered), R¹⁰⁴-substituted or unsubstituted arylene(e.g., C₆-C₁₀, C₁₀, or phenylene), or R¹⁰⁴-substituted or unsubstitutedheteroarylene (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

R¹⁰⁴ is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(104A)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-CC₁-C₆, or C₁-C₄), R^(104A)-substituted or unsubstituted heteroalkyl(e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),R^(104A)-substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), R^(104A)-substituted or unsubstituted heterocycloalkyl (e.g., 3to 8, 3 to 6, or 5 to 6 membered), R^(104A)-substituted or unsubstitutedaryl (e.g., C₆-C₁₀, C₁₀, or phenyl), or R^(104A)-substituted orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

R^(104A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(104B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(104B)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(104B)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(104B)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(104B)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(104B)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

R^(104B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, L¹⁰⁵ is a bond, —NH—, —NR¹⁰⁵—, —S—, —O—, —C(O)—,—C(O)O—, —OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—,R¹⁰⁵-substituted or unsubstituted alkylene (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R¹⁰⁵-substituted or unsubstitutedheteroalkylene (e.g., 2 to 20 membered, 8 to 20 membered, 5 to 16membered, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), R¹⁰⁵-substitutedor unsubstituted cycloalkylene (e.g., C₃-C₈, C₃-C₆, or C₅-C₆),R^(1o5)-substituted or unsubstituted heterocycloalkylene (e.g., 3 to 8,3 to 6, or 5 to 6 membered), R¹⁰⁵-substituted or unsubstituted arylene(e.g., C₆-C₁₀, C₁₀, or phenylene), or Rios-substituted or unsubstitutedheteroarylene (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

R¹⁰⁵ is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(105A)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(105A)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(105A)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(105A)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(105A)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(105A)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

R^(105A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(105B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(105B)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(105B)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(105B)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(105B)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(105B)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

R^(105B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,unsubstituted alkyl (e.g., C₁-C₁₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, L¹⁰¹, L¹⁰³, L¹⁰⁴, and L^(10n) are independently a bond,substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene.

In embodiments, L¹⁰¹ is a substituted or unsubstituted alkylene orsubstituted or unsubstituted 8 to 20 membered heteroalkylene; L¹⁰³ is abond or substituted or unsubstituted 2 to 10 membered heteroalkylene;L¹⁰⁴ is a bond, substituted or unsubstituted 4 to 18 memberedheteroalkylene, or substituted or unsubstituted phenylene; L^(10n) is abond or substituted or unsubstituted 4 to 18 membered heteroalkylene.

In embodiments, L¹⁰¹, L¹⁰³, and L^(10n) are independently a bond, —NH—,—O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—,substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene. In embodiments,L¹⁰⁴ is unsubstituted phenylene.

In embodiments, L¹⁰¹ is a substituted or unsubstituted C₁-C₄ alkylene orsubstituted or unsubstituted 8 to 20 membered heteroalkylene. Inembodiments, L¹⁰³ is a bond or substituted or unsubstituted 2 to 10membered heteroalkylene. In embodiments, L¹⁰⁴ is an unsubstitutedphenylene. In embodiments, L¹⁰⁵ is a bond or substituted orunsubstituted 4 to 18 membered heteroalkylene.

In embodiments, L¹⁰¹ is

In embodiments, L¹⁰¹ is

In embodiments, L¹⁰¹ is

In embodiments, L¹⁰¹ is

In embodiments, L¹⁰¹ is

In embodiments, L¹⁰¹ is

In embodiments, L¹⁰¹ is

In embodiments, L¹⁰¹ is

In embodiments, L¹⁰¹ is

In embodiments, L¹⁰¹ is

In embodiments, L¹⁰¹ is

In embodiments, L¹⁰¹ is —CCCH₂—. In embodiments, L¹⁰¹ is

In embodiments, L¹⁰¹ is

In embodiments, L¹⁰² is —OCH(CR⁵R⁶Si(R⁷)(R⁸)(R⁹))SS— and L¹⁰³ is —CH₂—;wherein R⁵, R⁶, R⁷, R⁸ and R⁹ are as described herein. In embodiments,L¹⁰² is —OCH(CR⁵R⁶Si(R⁷)(R⁸)(R⁹))SS— and L¹⁰³ is unsubstitutedphenylene; wherein R⁵, R⁶, R⁷, R⁸ and R⁹ are as described herein. Inembodiments, L¹⁰² is —OCH(CR⁵R⁶Si(R⁷)(R⁸)(R⁹))— wherein R⁵, R⁶, R⁷, R⁸and R⁹ are as described herein. In embodiments, L¹⁰² is—OCH(CR⁵R⁶Si(R⁷)(R⁸)(R⁹))— and L¹⁰³ is unsubstituted phenylene; whereinR⁵, R⁶, R⁷, R⁸ and R⁹ are as described herein.

In embodiments, L¹⁰⁰ is

wherein R⁵, R⁶, R⁷, R⁸ and R⁹ are as described herein.

In embodiments, L¹⁰⁰ is

wherein R⁵, R⁶, R⁷, R⁸ and R⁹ are as described herein. In embodiments,L¹⁰⁰ is

wherein R⁵, R⁶, R⁷, R⁸ and R⁹ are as described herein. In embodiments,L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

wherein R⁵, R⁶, R⁷, R⁸ and R⁹ are as described herein. In embodiments,L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

wherein R⁵, R⁶, R⁷, R⁸ and R⁹ are as described herein. In embodiments,L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

wherein R⁵, R⁶, R⁷, R⁸ and R⁹ are as described herein. In embodiments,L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

wherein R⁵, R⁶, R⁷, R⁸ and R⁹ are as described herein. In embodiments,L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

wherein R⁵, R⁶, R⁷, R⁸ and R⁹ are as described herein. In embodiments,L¹⁰⁰ is

wherein R⁵, R⁶, R⁷, R⁸ and R⁹ are as described herein.

In embodiments, the compound has the formula:

wherein R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are as described herein; L¹⁰⁰ is acleavable linker; and R⁴ is a detectable moiety. In embodiments, L¹⁰⁰ is

wherein R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, L¹⁰³, L¹⁰⁴, L¹⁰⁵ are as describedherein. In embodiments, L¹⁰⁰ is

wherein L⁰³, L¹⁰⁴, L¹⁰⁵ are as described herein. In embodiments, L¹⁰⁰ is

wherein L¹⁰³, L¹⁰⁴, L¹⁰⁵ are as described herein. In embodiments, L¹⁰⁰is

wherein L¹⁰³, L¹⁰⁴, L¹⁰⁵ are as described herein.

In embodiments, L¹⁰⁰ is

In embodiments, the compound has the formula:

wherein L¹⁰⁰, R⁴, and R¹⁰ are as described herein.

In embodiments, the compound has the formula:

wherein L¹⁰⁰, R⁴, and R¹⁰ are as described herein.

In embodiments, the compound has the formula:

wherein L¹⁰⁰, R⁴, and R¹⁰ are as described herein.

In embodiments, R⁴ is a detectable moiety. In embodiments, R⁴ is afluorescent dye moiety. In embodiments, R⁴ is a detectable moietydescribed herein (e.g., a dye identified within Table 1). Inembodiments, R⁴ is a detectable moiety described in Table 1. Inembodiments, R4 is a monovalent Bodipy® 493/503, monovalentaminomethylcoumarin (AMCA), monovalent ANT, monovalent MANT, monovalentAmNS, monovalent 7-diethylaminocoumarin-3-carboxylic acid (DEAC),monovalent ATTO 390, monovalent Alexa Fluor® 350, monovalent MarinaBlue, monovalent Cascade Blue, or monovalent Pacific Blue. Inembodiments, the R⁴ is a fluorescent molecule (e.g., acridine dye,cyanine, dye, fluorine dye, oxazine dye, phenanthridine dye, orrhodamine dye).

In embodiments, R⁴ is a fluorescent dye moiety wherein the maximumemission of the fluorescent dye moiety is greater than about 530, 540,or 550 nm. In embodiments, R⁴ is a fluorescent dye moiety wherein themaximum emission of the fluorescent dye moiety is greater than 530 nm.In embodiments, R⁴ is a fluorescent dye moiety wherein the maximumemission of the fluorescent dye moiety is less than about 700, 690, or680 nm. In embodiments, R⁴ is a fluorescent dye moiety wherein themaximum emission of the fluorescent dye moiety is less than 680 nm. Inembodiments, R⁴ is a fluorescent dye moiety wherein the maximum emissionof the fluorescent dye moiety is greater than about 530 and less thanabout 680 nm. In embodiments, R⁴ is a fluorescent dye moiety wherein themaximum emission of the fluorescent dye moiety is greater than 530 andless than 680 nm. For example, R⁴ may be any fluorescent moietydescribed in US Publication 2020/0216682, which is incorporated hereinby reference.

TABLE 1 Detectable moieties to be used in selected embodiments.Nucleoside/nucleotide abbreviation Dye name λmax (nm) dC Atto 532 532 dCAtto Rho 6G 535 dC R6G 534 dC Tet 521 dT Atto Rho 11 572 dT Atto 565 564dT Alexa Fluor 568 578 dT dTamra 578 dA Alexa Fluor 647 650 dA Atto 647N644 dA Janelia Fluor 646 646 dG Alexa Fluor 680 682 dG Alexa Fluor 700696 dG CF680R 680

In embodiments, R⁴ is

In embodiments, R⁴ is a quenching moiety. In embodiments, R⁴ is aquencher. The quencher may provide an additional benefit by quenching(i.e., absorbing) any remaining fluorescence before the next sequencingcycle. For example, quenching moieties reduce signal cross-talk therebysimplifying nucleotide detection. Non-limiting examples of quenchingmoieties include monovalent species of Dabsyl(dimethylaminoazobenzenesulfonic acid), Black Hole Quenchers (BHQ)(e.g., (BHQ), BHQ-2, and BHQ-3), BMN Quenchers (e.g., BMN-Q460,BMN-Q535, BMN-Q590, BMN-Q620, BMN-Q650) Qxl, Tide Quenchers (e.g., TQ2,TQ3), Iowa black FQ, Iowa black RQ, Deep Dark Quencher (e.g., DDQ I, DDQII), or IRDye QC-1. In embodiments, R⁴ is BMN-Q460, Dabcyl, DDQ-I,BMN-Q535, HHQ-1, TQ2, BMN-Q620, BMN-Q590, BHQ-2, TQ3, BMN-Q650, orBBQ-650. In embodiments, R⁴ is a quenching moiety capable of quenchingfluorescence in range of 400-530 nm, 480-580 nm, 550-650 nm, 480-720 nm,or 550-720 nm.

In an aspect is provided a nucleic acid polymerase complex including anucleic acid polymerase, wherein the nucleic acid polymerase is bound toa compound as described herein (e.g., a compound of Formula I or FormulaII) and in related embodiments. In embodiments, the complex is furtherbound to a primer, wherein the primer is hybridized to a templatepolynucleotide.

In embodiments, the nucleic acid polymerase is a Taq polymerase,Therminator γ, 9° N polymerase (exo-), Therminator II, Therminator III,or Therminator IX. In embodiments, the nucleic acid polymerase isTherminator γ. In embodiments, the nucleic acid polymerase is 9° Npolymerase (exo-). In embodiments, the nucleic acid polymerase isTherminator II. In embodiments, the nucleic acid polymerase isTherminator III. In embodiments, the nucleic acid polymerase isTherminator IX. In embodiments, the nucleic acid polymerase is a Taqpolymerase. In embodiments, the nucleic acid polymerase is a nucleicacid polymerase. In embodiments, the nucleic acid polymerase is 9° N andmutants thereof. In embodiments, the nucleic acid polymerase is Phi29and mutants thereof. In embodiments, the DNA polymerase is a modifiedarchaeal DNA polymerase. In embodiments, the polymerase is a reversetranscriptase. In embodiments, the polymerase is a mutant P. abyssipolymerase (e.g., such as a mutant P. abyssi polymerase described in WO2018/148723 or WO 2020/056044). Other useful DNA polymerases includethermostable and/or thermophilic DNA polymerases such as Thermusaquaticus (Taq) DNA polymerase, Thermus filiformis (Tfi) DNA polymerase,Thermococcus zilligi (Tzi) DNA polymerase, Thermus thermophilus (Tth)DNA polymerase, Thermus flavusu (Tfl) DNA polymerase, Pyrococcus woesei(Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase and TurboPfu DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase,Pyrococcus sp. GB-D polymerase, Thermotoga maritima (Tma) DNApolymerase, Bacillus stearothermophilus (Bst) DNA polymerase, PyrococcusKodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase, Thermococcus sp.JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius (Tgo) DNApolymerase, Thermococcus acidophilium DNA polymerase; Sulfolobusacidocaldarius DNA polymerase; Thermococcus sp. go N-7 DNA polymerase;Pyrodictium occultum DNA polymerase; Methanococcus voltae DNApolymerase; Methanococcus thermoautotrophicum DNA polymerase;Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNApolymerase (D. Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcushorikoshii DNA polymerase; Pyrococcus islandicum DNA polymerase;Thermococcus fumicolans DNA polymerase; Aeropyrum pernix DNA polymerase;and the heterodimeric DNA polymerase DP1/DP2. In embodiments, thepolymerase is 3PDX polymerase as disclosed in U.S. Pat. No. 8,703,461,the disclosure of which is incorporated herein by reference. Inembodiments, the polymerase is a reverse transcriptase. Exemplaryreverse transcriptases include, but are not limited to, HIV-1 reversetranscriptase from human immunodeficiency virus type 1 (PDB 1HMV), HIV-2reverse transcriptase from human immunodeficiency virus type 2, M-MLVreverse transcriptase from the Moloney murine leukemia virus, AMVreverse transcriptase from the avian myeloblastosis virus, or Telomerasereverse transcriptase. In embodiments, the depletion polymerase is anucleotide cyclase. In embodiments, the depletion polymerase is aterminal transferase (e.g., terminal deoxycytidyl transferase orterminal deoxynucleotidyl transferase (TdT)). In embodiments, thedepletion polymerase is an RNA dependent polymerase. In embodiments, thedepletion polymerase is a Klenow fragment or mutant thereof, solubleguanylyl cyclase or mutant thereof, or a terminal deoxynucleotidyltransferase (TdT).

In an aspect is provided a kit. Some embodiments disclosed herein relateto kits including a labeled nucleoside or nucleotide (e.g., a compoundas described herein) including a linker between the fluorophore and thenucleoside or nucleotide, wherein the linker is a linker as describedherein. In embodiments, the kit includes a compound described herein. Inembodiments, the kit includes a plurality of compounds described herein.In embodiments, the kit includes labeled nucleotides includingdifferently labeled nucleotides (e.g., compounds described herein). Inembodiments, the kit further includes instructions for use thereof. Inembodiments, kits described herein include a polymerase. In embodiments,the polymerase is a DNA polymerase. In embodiments, the DNA polymeraseis a thermophilic nucleic acid polymerase. In embodiments, the DNApolymerase is a modified archaeal DNA polymerase. In embodiments, thekit includes a sequencing solution. In embodiments, the sequencingsolution include labeled nucleotides including differently labelednucleotides, wherein the label (or lack thereof) identifies the type ofnucleotide. For example, each adenine nucleotide, or analog thereof athymine nucleotide; a cytosine nucleotide, or analog thereof; and aguanine nucleotide, or analog thereof may be labeled with a differentfluorescent label.

In embodiments, the sequencing solution includes a buffer solution.Typically, the buffered solutions contemplated herein are made from aweak acid and its conjugate base or a weak base and its conjugate acid.For example, sodium acetate and acetic acid are buffer agents that canbe used to form an acetate buffer. Other examples of buffer agents thatcan be used to make buffered solutions include, but are not limited to,Tris, Tricine, HEPES, TES, MOPS, MOPSO and PIPES. In embodiments, thebuffer includes ethanolamine (EA), tris(hydroxymethyl)aminomethane(Tris), glycine, a carbonate salt, a phosphate salt, a borate salt,2-dimethyalaminomethanol (DMEA), 2-diethyalaminomethanol (DEEA),N,N,N′,N′-tetramethylethylenediamine (TEMED), andN,N,N′,N′-tetraethylethylenediamine (TEEDA), or a combination thereof.Additionally, other buffer agents that can be used in enzyme reactions,hybridization reactions, and detection reactions are well known in theart. In embodiments, the buffered solution can include Tris. Withrespect to the embodiments described herein, the pH of the bufferedsolution can be modulated to permit any of the described reactions. Insome embodiments, the buffered solution can have a pH greater than pH7.0, greater than pH 7.5, greater than pH 8.0, greater than pH 8.5,greater than pH 9.0, greater than pH 9.5, greater than pH 10, greaterthan pH 10.5, greater than pH 11.0, or greater than pH 11.5. In otherembodiments, the buffered solution can have a pH ranging, for example,from about pH 6 to about pH 9, from about pH 8 to about pH 10, or fromabout pH 7 to about pH 9. In embodiments, the buffered solution cancomprise one or more divalent cations. Examples of divalent cations caninclude, but are not limited to, Mg²⁺, Mn²⁺, Zn²⁺, and Ca²⁺. Inembodiments, the buffered solution can contain one or more divalentcations at a concentration sufficient to permit hybridization of anucleic acid. In some embodiments, a concentration can be more thanabout 1 μM, more than about 2 μM, more than about 5 μM, more than about10 μM, more than about 25 μM, more than about 50 μM, more than about 75μM, more than about 100 μM, more than about 200 μM, more than about 300μM, more than about 400 μM, more than about 500 μM, more than about 750μM, more than about 1 mM, more than about 2 mM, more than about 5 mM,more than about 10 mM, more than about 20 mM, more than about 30 mM,more than about 40 mM, more than about 50 mM, more than about 60 mM,more than about 70 mM, more than about 80 mM, more than about 90 mM,more than about 100 mM, more than about 150 mM, more than about 200 mM,more than about 250 mM, more than about 300 mM, more than about 350 mM,more than about 400 mM, more than about 450 mM, more than about 500 mM,more than about 550 mM, more than about 600 mM, more than about 650 mM,more than about 700 mM, more than about 750 mM, more than about 800 mM,more than about 850 mM, more than about 900 mM, more than about 950 mMor more than about 1 M.

In embodiments, the kit includes nucleotides in a buffer. Inembodiments, the kit includes a buffer. For example, the sequencingsolution and/or the chase solution may include a buffer such asethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, acarbonate salt, a phosphate salt, a borate salt,2-dimethyalaminomethanol (DMEA), 2-diethyalaminomethanol (DEEA),N,N,N′,N′-tetramethylethylenediamine (TEMED), andN,N,N′,N′-tetraethylethylenediamine (TEEDA), and combinations thereof.For example, the buffer may Tris-HCl (pH 9.2 at 25° C.), ammoniumsulfate, MgCl₂, 0.1% Tween® 20, and dNTPs.

In embodiments, the kit includes a solid support (e.g., a flow cell ormicroplate). Flow cells provide a convenient format for housing an arrayof clusters produced by the methods described herein, in particular whensubjected to an SBS or other detection technique that involves repeateddelivery of reagents in cycles. For example, to initiate a first SBScycle, one or more labeled nucleotides and a DNA polymerase in a buffercan be flowed into/through a flow cell that houses an array of clusters.The clusters of an array where primer extension causes a labelednucleotide to be incorporated can then be detected. Optionally, thenucleotides can further include a reversible termination moiety thattemporarily halts further primer extension once a nucleotide has beenadded to a primer. For example, a nucleotide analog having a reversibleterminator moiety can be added to a primer such that subsequentextension cannot occur until a deblocking agent (e.g., a reducing agent)is delivered to remove the moiety. Thus, for embodiments that usereversible termination, a deblocking reagent (e.g., a reducing agent)can be delivered to the flow cell (before, during, or after detectionoccurs). Washes can be carried out between the various delivery steps asneeded. The cycle can then be repeated N times to extend the primer by Nnucleotides, thereby detecting a sequence of length N. Example SBSprocedures, fluidic systems and detection platforms that can be readilyadapted for use with an array produced by the methods of the presentdisclosure are described, for example, in Bentley et al., Nature456:53-59 (2008).

In embodiments, the kit includes a plurality of primers for amplifyingand/or for sequencing nucleic acids isolated from the sample. The kitmay provide at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200,500, 1000, or more primers. The kit may provide between about 1-3, 1-10,5-20, 1-1000, 10-500, 20-200, or 50-100 primers. In embodiments, theprimers include 5, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200 or morenucleotides.

In embodiments, the kit includes a buffer. In embodiments, the kitincludes a buffered solution. For example, the sequencing solutionand/or the chase solution may include a buffer such as ethanolamine(EA), tris(hydroxymethyl)aminomethane (Tris), glycine, a carbonate salt,a phosphate salt, a borate salt, 2-dimethyalaminomethanol (DMEA),2-diethyalaminomethanol (DEEA), N,N,N′,N′-tetramethylethylenediamine(TEMED), and N,N,N′,N′-tetraethylethylenediamine (TEEDA), andcombinations thereof. Typically, the buffered solutions contemplatedherein are made from a weak acid and its conjugate base or a weak baseand its conjugate acid. For example, sodium acetate and acetic acid arebuffer agents that can be used to form an acetate buffer. Other examplesof buffer agents that can be used to make buffered solutions include,but are not limited to, Tris, Bicine, Tricine, HEPES, TES, MOPS, MOPSOand PIPES. Additionally, other buffer agents that can be used in enzymereactions, hybridization reactions, and detection reactions are known inthe art. In embodiments, the buffered solution can include Tris. Withrespect to the embodiments described herein, the pH of the bufferedsolution can be modulated to permit any of the described reactions. Insome embodiments, the buffered solution can have a pH greater than pH7.0, greater than pH 7.5, greater than pH 8.0, greater than pH 8.5,greater than pH 9.0, greater than pH 9.5, greater than pH 10, greaterthan pH 10.5, greater than pH 11.0, or greater than pH 11.5. In otherembodiments, the buffered solution can have a pH ranging, for example,from about pH 6 to about pH 9, from about pH 8 to about pH 10, or fromabout pH 7 to about pH 9. In embodiments, the buffered solution cancomprise one or more divalent cations. Examples of divalent cations caninclude, but are not limited to, Mg²⁺, Mn²⁺, Zn²⁺, and Ca²⁺. Inembodiments, the buffered solution can contain one or more divalentcations at a concentration sufficient to permit hybridization of anucleic acid. In embodiments, the buffer includes PEG (polyethyleneglycol), PVP (polyvinylpyrrolidone), trehalose, ficoll, or dextran. Inembodiments, the buffer includes additives such as Tween-20 or NP-40.

III. Methods

In an aspect is provided a method for sequencing a nucleic acid. Inembodiments, the method includes, (i) incorporating in series with anucleic acid polymerase, (e.g., within a reaction vessel) one of fourdifferent compounds (e.g., nucleotide analogues) into a primer to createan extension strand, wherein the primer is hybridized to the nucleicacid and wherein each of the four different compounds comprises a uniquedetectable label; and (ii) detecting the unique detectable label of eachincorporated compound, so as to thereby identify each incorporatedcompound in the extension strand, thereby sequencing the nucleic acid;wherein each of the four different compounds is independently a compoundas described herein, including embodiments. In embodiments, the methodincludes cleaving the linker (e.g., cleaving) L¹⁰⁰. In embodiments,cleaving the linker includes contacting the compound with a reducingagent (e.g., tris(3-hydroxypropyl)phosphine). In embodiments, the methodincludes removing (e.g., cleaving) the reversible terminator moiety. Inembodiments, the method includes removing (e.g., cleaving) R³ togenerate a 3′-OH. In embodiments, the method includes chemicallycleaving the linker and/or the polymerase-compatible cleavable moiety asdescribed herein (e.g., L¹⁰⁰ and/or R³ of the compounds of Formula I andFormula II).

A variety of sequencing methodologies can be used such as sequencing-bysynthesis (SBS), sequencing by ligation (SBL), or sequencing byhybridization (SBH). In both SBL and SBH methods, target nucleic acids,and amplicons thereof, that are present at features of an array aresubjected to repeated cycles of oligonucleotide delivery and detection.SBL methods, include those described in Shendure et al. Science309:1728-1732 (2005); U.S. Pat. Nos. 5,599,675; and 5,750,341, each ofwhich is incorporated herein by reference in its entirety; and the SBHmethodologies are as described in Bains et al., Journal of TheoreticalBiology 135(3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16,54-58 (1998); Fodor et al., Science 251(4995), 767-773 (1995); and WO1989/10977, each of which is incorporated herein by reference in itsentirety.

In SBS, extension of a nucleic acid primer along a nucleic acid templateis monitored to determine the sequence of nucleotides in the template.The underlying chemical process can be catalyzed by a polymerase,wherein fluorescently labeled nucleotides are added to a primer (therebyextending the primer) in a template dependent fashion such thatdetection of the order and type of nucleotides added to the primer canbe used to determine the sequence of the template. A plurality ofdifferent nucleic acid fragments that have been attached at differentlocations of an array can be subjected to an SBS technique underconditions where events occurring for different templates can bedistinguished due to their location in the array. In embodiments, thesequencing step includes annealing and extending a sequencing primer toincorporate a detectable label that indicates the identity of anucleotide in the target polynucleotide, detecting the detectable label,and repeating the extending and detecting of steps. In embodiments, themethods include sequencing one or more bases of a target nucleic acid byextending a sequencing primer hybridized to a target nucleic acid (e.g.,an amplification product produced by the amplification methods describedherein). In embodiments, the sequencing step may be accomplished by asequencing-by-synthesis (SBS) process. In embodiments, sequencingincludes a sequencing by synthesis process, where individual nucleotidesare identified iteratively, as they are polymerized to form a growingcomplementary strand. In embodiments, nucleotides added to a growingcomplementary strand include both a label and a reversible chainterminator that prevents further extension, such that the nucleotide maybe identified by the label before removing the terminator to add andidentify a further nucleotide. Such reversible chain terminators includeremovable 3′ blocking groups, alternatively referred to as reversibleterminators or polymerase-compatible cleavable moieties as describedherein, for example as described in U.S. Pat. Nos. 10,738,072,10,822,653, and 11,174,281. Once such a modified nucleotide has beenincorporated into the growing polynucleotide chain complementary to theregion of the template being sequenced, there is no free 3′—OH groupavailable to direct further sequence extension and therefore thepolymerase cannot add further nucleotides. Once the identity of the baseincorporated into the growing chain has been determined, the 3′ blockmay be removed to allow addition of the next successive nucleotide. Byordering the products derived using these modified nucleotides it ispossible to deduce the DNA sequence of the DNA template (e.g., byobtaining a sequencing read).

In embodiments, chemical cleavage of a compound (e.g., cleavage of the3′ moiety of a compound described herein, cleavage of a linker (e.g., alinker including L¹⁰⁰) as described herein, or cleavage of an SS bond ina 3′ moiety of a compound described herein) described herein (e.g., inan aspect or embodiment) includes contacting the compound with areducing agent (e.g., tris(hydroxypropyl)phosphine (THPP),tris-(2-carboxyethyl)phosphine (TCEP), tris(hydroxymethyl)phosphine(THMP), or tris(hydroxyethyl)phosphine (THEP), DTT, dithiobutylamine(DTBA)). In embodiments, the chemical cleavage may include one or morecatalysts (e.g., Pd(0)). In embodiments, chemical cleavage of a compound(e.g., cleavage of the 3′ moiety of a compound described herein,cleavage of a linker (e.g., a linker including L¹⁰⁰) as describedherein, or cleavage of an SS bond in a 3′ moiety of a compound describedherein) described herein (e.g., in an aspect or embodiment) includescontacting the compound with THPP (e.g., about 10 mM THPP, or at least 1mM THPP). In embodiments, chemical cleavage of a compound (e.g.,cleavage of the 3′ moiety of a compound described herein, cleavage of alinker (e.g., a linker including L¹⁰⁰) as described herein, or cleavageof an SS bond in a 3′ moiety of a compound described herein) describedherein (e.g., in an aspect or embodiment) is performed at less thanabout 65° C. In embodiments, chemical cleavage of a compound (e.g.,cleavage of the 3′ moiety of a compound described herein, cleavage of alinker (e.g., a linker including L¹⁰⁰) as described herein, or cleavageof an SS bond in a 3′ moiety of a compound described herein) describedherein (e.g., in an aspect or embodiment) is performed at less than 65°C. In embodiments, chemical cleavage of a compound (e.g., cleavage ofthe 3′ moiety of a compound described herein, cleavage of a linker(e.g., a linker including L¹⁰⁰) as described herein, or cleavage of anSS bond in a 3′ moiety of a compound described herein) described herein(e.g., in an aspect or embodiment) is performed at about 45-65° C. Inembodiments, chemical cleavage of a compound (e.g., cleavage of the 3′moiety of a compound described herein, cleavage of a linker (e.g., alinker including L¹⁰⁰) as described herein, or cleavage of an SS bond ina 3′ moiety of a compound described herein) described herein (e.g., inan aspect or embodiment) is performed at 45-65° C. In embodiments,chemical cleavage of a compound (e.g., cleavage of the 3′ moiety of acompound described herein, cleavage of a linker (e.g., a linkerincluding L¹⁰⁰) as described herein, or cleavage of an SS bond in a 3′moiety of a compound described herein) described herein (e.g., in anaspect or embodiment) is performed at 45° C., 46° C., 47° C., 48° C.,49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C.,58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., or 65° C. Inembodiments, chemical cleavage of a compound (e.g., cleavage of the 3′moiety of a compound described herein, cleavage of a linker (e.g., alinker including L¹⁰⁰) as described herein, or cleavage of an SS bond ina 3′ moiety of a compound described herein) described herein (e.g., inan aspect or embodiment) is performed at about 55° C. In embodiments,chemical cleavage of a compound (e.g., cleavage of the 3′ moiety of acompound described herein, cleavage of a linker (e.g., a linkerincluding L¹⁰⁰) as described herein, or cleavage of an SS bond in a 3′moiety of a compound described herein) described herein (e.g., in anaspect or embodiment) is performed at a temperature of at least 55° C.In embodiments, chemical cleavage of a compound (e.g., cleavage of the3′ moiety of a compound described herein, cleavage of a linker (e.g., alinker including L¹⁰⁰) as described herein, or cleavage of an SS bond ina 3′ moiety of a compound described herein) described herein (e.g., inan aspect or embodiment) is performed at about pH 9.5. In embodiments,chemical cleavage of a compound (e.g., cleavage of the 3′ moiety of acompound described herein, cleavage of a linker (e.g., a linkerincluding L¹⁰⁰) as described herein, or cleavage of an SS bond in a 3′moiety of a compound described herein) described herein (e.g., in anaspect or embodiment) is performed at about pH 9.5. In embodiments,chemical cleavage of a compound (e.g., cleavage of the 3′ moiety of acompound described herein, cleavage of a linker (e.g., a linkerincluding L¹⁰⁰) as described herein, or cleavage of an SS bond in a 3′moiety of a compound described herein) described herein (e.g., in anaspect or embodiment) is performed at pH 9.5. In embodiments, chemicalcleavage of a compound (e.g., cleavage of the 3′ moiety of a compounddescribed herein, cleavage of a linker (e.g., a linker including L¹⁰⁰)as described herein, or cleavage of an SS bond in a 3′ moiety of acompound described herein) described herein (e.g., in an aspect orembodiment) is performed using 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, or 1.0 mM of THPP. In embodiments, the chemical cleavage isperformed using less than 1.0 mM THPP. In embodiments, the chemicalcleavage is performed using about 1.0 mM THPP. In embodiments, thechemical cleavage is performed using about 0.05 to about 1.0 mM THPP. Inembodiments, the chemical cleavage is performed using about 1.0 to about5.0 mM THPP. In embodiments, the chemical cleavage is performed usingabout 10 mM THPP. In embodiments, the chemical cleavage is performedusing 1.0 mM THPP. In embodiments, the chemical cleavage is performedusing about 0.05 to 1.0 mM THPP. In embodiments, the chemical cleavageis performed using 1.0 to about 5.0 mM THPP. In embodiments, thechemical cleavage is performed using 10 mM THPP.

A number of new techniques have been described for reading out RNAtranscription levels in tissue sections directly (i.e., in-situ),without requiring spatial barcoding, based on single moleculefluorescence in situ hybridization. These include MERFISH (MultiplexedError-Robust Fluorescence In Situ Hybridization), STARmap(Spatially-resolved Transcript Amplicon Readout mapping), DART-FISH,seq-FISH (Sequential Fluorescence In Situ Hybridization), FISSEQ(fluorescent in situ sequencing), and others (see for example Chen, K.H., et al. (2015). Science, 348(6233), aaa6090; Wang, G., Moffitt, J. R.& Zhuang, X. Sci Rep. 2018; 8, 4847; Wang X. et al; Science, 2018; 27,Vol 361, Issue 6400, eaat5691; Cai, M. Dissertation, (2019) UC SanDiego. ProQuest ID: Cai_ucsd_0033D_18822; Lee J H et al. Nat. Protoc.2015; 10(3):442-58); and Sansone, A. Nat Methods 16, 458; 2019). In allof these techniques, individual RNA transcripts are individuallyresolved, typically with pre-amplification or requiring multipleinstances of labeled probes. Some of these techniques have been combinedwith super-resolution microscopy, expansion microscopy, or both, toincrease the resolution and allow more transcripts to be resolved andthus counted.

In embodiments, the compounds are described herein. In embodiments, thefour different compounds are labeled nucleotide analogues as describedherein (e.g., four different compounds described herein each including adifferent nucleobase and a different label (e.g., fluorescent dyemoiety)). In embodiments, the four different labeled nucleotideanalogues are four different compounds described herein (e.g., fourdifferent compounds described herein each including a differentnucleobase). In embodiments, the four different labeled nucleotideanalogues are four different compounds described herein (e.g., fourdifferent compounds described herein each including a different label(e.g., fluorescent dye moiety)).

In embodiments, the method further including, after each of theincorporating steps, adding to the reaction vessel four differentunlabeled nucleotide analogues, wherein each of the four differentunlabeled nucleotide analogues are of the structure as described herein,including embodiments, wherein in the first of the four differentunlabeled nucleotide analogues, B is a thymidine or uridine hybridizingbase; in the second of the four different unlabeled nucleotideanalogues, B is an adenosine hybridizing base; in the third of the fourdifferent unlabeled nucleotide analogues, B is a guanosine hybridizingbase; and in the fourth of the four different unlabeled nucleotideanalogues, B is a cytosine hybridizing base.

In an aspect is provided a method for sequencing a nucleic acid,including (i) incorporating in series with a nucleic acid polymerase,within a reaction vessel, one of four different compounds into a primerto create an extension strand, wherein the primer is hybridized to thenucleic acid and wherein each of the four different compounds includes aunique detectable label; (ii) detecting the unique detectable label ofeach incorporated compound, so as to thereby identify each incorporatedcompound in the extension strand, thereby sequencing the nucleic acid;wherein each of the four different compounds is independently a compoundas described herein, including embodiments. In embodiments, the compoundincludes at least one of the following: cytosine or a derivativethereof, guanine or a derivative thereof, adenine or a derivativethereof, thymine or a derivative thereof, uracil or a derivativethereof, hypoxanthine or a derivative thereof, xanthine or a derivativethereof, 7-methylguanine or a derivative thereof, 5,6-dihydrouracil or aderivative thereof, 5-methylcytosine or a derivative thereof, and5-hydroxymethylcytosine or a derivative thereof. In embodiments, thecompound includes at least one of the following: cytosine or aderivative thereof, guanine or a derivative thereof, adenine or aderivative thereof, thymine or a derivative thereof, and uracil or aderivative thereof. In embodiments, the compound includes at least oneof the following: cytosine or a derivative thereof, guanine or aderivative thereof, adenine or a derivative thereof, and thymine or aderivative thereof. In embodiments, the compound includes at least oneof the following: cytosine or a derivative thereof, guanine or aderivative thereof, adenine or a derivative thereof, and uracil or aderivative thereof. In embodiments, the method further includes, afterincorporating, contacting the compound with a cleaving agent. Inembodiments, the method includes incorporating a first nucleotideincluding a 3′-O-reversible terminator and a first detectable label;detecting the first detectable label; and removing the 3′-O-reversibleterminator from the first nucleotide to generate a nucleotide includinga 3′-OH. In embodiments, the method includes generating one or moresequencing reads.

In embodiments, the nucleic acid to be sequenced is DNA or RNA, or ahybrid molecule comprised of deoxynucleotides and ribonucleotides. Inembodiments, the nucleic acid to be sequenced is attached to a solidsubstrate via any suitable linkage method known in the art, e.g., usingcovalent linkage. In embodiments, the nucleic acid is attached directlyto a solid substrate. In embodiments, the surface of the solid supportincludes a polymer that provides the attachment points for the nucleicacid.

In embodiments, the nucleic acid is within a cluster. The terms“cluster” and “colony” are used interchangeably throughout thisapplication and refer to a discrete site on a solid support comprised ofa plurality of immobilized nucleic acid strands. The term “clusteredarray” refers to an array formed from such clusters or colonies. In thiscontext the term “array” is not to be understood as requiring an orderedarrangement of clusters. The term “array” is used in accordance with itsordinary meaning in the art, and refers to a population of differentmolecules that are attached to one or more solid-phase substrates suchthat the different molecules can be differentiated from each otheraccording to their relative location. An array can include differentmolecules that are each located at different addressable features on asolid-phase substrate. The molecules of the array can be nucleic acidprimers, nucleic acid probes, nucleic acid templates or nucleic acidenzymes such as polymerases or ligases. Arrays useful in the inventioncan have densities that ranges from about 2 different features to manymillions, billions or higher. The density of an array can be from 2 toas many as a billion or more different features per square cm. Forexample an array can have at least about 100 features/cm², at leastabout 1,000 features/cm², at least about 10,000 features/cm², at leastabout 100,000 features/cm², at least about 10,000,000 features/cm², atleast about 100,000,000 features/cm², at least about 1,000,000,000features/cm², at least about 2,000,000,000 features/cm² or higher. Inembodiments, the arrays have features at any of a variety of densitiesincluding, for example, at least about 10 features/cm², 100features/cm², 500 features/cm², 1,000 features/cm², 5,000 features/cm²,10,000 features/cm², 50,000 features/cm², 100,000 features/cm²,1,000,000 features/cm², 5,000,000 features/cm², or higher.

In another aspect is provided a method of incorporating a compound intoa primer, the method including combining a polymerase, a primerhybridized to nucleic acid template and the compound within a reactionvessel and allowing the polymerase to incorporate the compound into theprimer thereby forming an extended primer, wherein the compound is acompound as described herein, including embodiments. In embodiments,incorporating a compound into a primer refers to the 5′ phosphatejoining in phosphodiester linkage to the 3′—OH group of a second(modified or unmodified) nucleotide, which may itself form part of alonger polynucleotide chain.

In embodiments, the method further including, after the incorporating,cleaving the linker (e.g., L¹⁰⁰) with a cleaving reagent (e.g.,tris(hydroxypropyl)phosphine (THPP)). In embodiments, the cleavingreagent is an acid, base, oxidizing agent, reducing agent, Pd(0),tris-(2-carboxyethyl)phosphine, dilute nitrous acid, fluoride,tris(3-hydroxypropyl)phosphine), sodium dithionite (Na₂S₂O₄), orhydrazine (N₂H₄). In embodiments, the cleaving reagent is in a buffer.In embodiments, the buffer includes an acetate buffer,3-(N-morpholino)propanesulfonic acid (MOPS) buffer,N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES) buffer,phosphate-buffered saline (PBS) buffer,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer,N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO) buffer, borate buffer (e.g., borate buffered saline, sodiumborate buffer, boric acid buffer), 2-Amino-2-methyl-1,3-propanediol(AMPD) buffer, N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid(CAPSO) buffer, 2-Amino-2-methyl-1-propanol (AMP) buffer,4-(Cyclohexylamino)-1-butanesulfonic acid (CABS) buffer, glycine-NaOHbuffer, N-Cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer,tris(hydroxymethyl)aminomethane (Tris) buffer, or aN-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer. In embodiments,the buffer is a borate buffer. In embodiments, the buffer is a CHESbuffer. In embodiments, the method includes contacting the compound(e.g., a compound described herein) with a reducing agent. Inembodiments, the method further including, after the incorporating,cleaving the linker at about 55° C. In embodiments, the method furtherincluding, after the incorporating, cleaving the linker at about 55° C.to about 80° C. In embodiments, the method further including, after theincorporating, cleaving the linker at about 60° C. to about 70° C. Inembodiments, the method further including, after the incorporating,cleaving the linker at about 65° C. to about 75° C. In embodiments, themethod further including, after the incorporating, cleaving the linkerat about 65° C. In embodiments, the method further including, after theincorporating, cleaving the linker at about 55° C., 56° C., 57° C., 58°C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67°C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76°C., 77° C., 78° C., 79° C., or about 80° C. In embodiments, the methodfurther including, after the incorporating, cleaving the linker at a pHat about 8.0 to 11.0. In embodiments, the pH is 9.0 to 11.0. Inembodiments, the pH is 9.5. In embodiments, the pH is 10.0. Inembodiments, the pH is 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, or 11.0. Inembodiments, the pH is from 9.0 to 11.0, and the temperature is fromabout 60° C. to about 70° C.

In embodiments, the cleaving reagent cleaves both the linker and thepolymerase-compatible cleavable moiety simultaneously.

In embodiments, the nucleic acid polymerase is a Taq polymerase,Therminator γ, 9° N polymerase (exo-), Therminator II, Therminator III,or Therminator IX. In embodiments, the thermophilic nucleic acidpolymerase is Therminator γ. In embodiments, the thermophilic nucleicacid polymerase is 9° N polymerase (exo-). In embodiments, thethermophilic nucleic acid polymerase is Therminator II. In embodiments,the thermophilic nucleic acid polymerase is Therminator III. Inembodiments, the thermophilic nucleic acid polymerase is Therminator IX.In embodiments, the thermophilic nucleic acid polymerase is a Taqpolymerase. In embodiments, the nucleic acid polymerase is athermophilic nucleic acid polymerase. In embodiments, the nucleic acidpolymerase is 9° N and mutants thereof. In embodiments, the nucleic acidpolymerase is Phi29 and mutants thereof. In embodiments, the polymeraseis a non-thermophilic nucleic acid polymerase. Other useful DNApolymerases include thermostable and/or thermophilic DNA polymerasessuch as Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis (Tfi)DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermusthermophilus (Tth) DNA polymerase, Thermus flavusu (Tfl) DNA polymerase,Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNApolymerase and Turbo Pfu DNA polymerase, Thermococcus litoralis (Tli)DNA polymerase, Pyrococcus sp. GB-D polymerase, Thermotoga maritima(Tma) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase,Pyrococcus Kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase,Thermococcus sp. JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius(Tgo) DNA polymerase, Thermococcus acidophilium DNA polymerase;Sulfolobus acidocaldarius DNA polymerase; Thermococcus sp. go N-7 DNApolymerase; Pyrodictium occultum DNA polymerase; Methanococcus voltaeDNA polymerase; Methanococcus thermoautotrophicum DNA polymerase;Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNApolymerase (D. Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcushorikoshii DNA polymerase; Pyrococcus islandicum DNA polymerase;Thermococcus fumicolans DNA polymerase; Aeropyrum pernix DNA polymerase;and the heterodimeric DNA polymerase DP1/DP2. In embodiments, thepolymerase is 3PDX polymerase as disclosed in U.S. Pat. No. 8,703,461,the disclosure of which is incorporated herein by reference. Inembodiments, the polymerase is a reverse transcriptase. Exemplaryreverse transcriptases include, but are not limited to, HIV-1 reversetranscriptase from human immunodeficiency virus type 1 (PDB 1HMV), HIV-2reverse transcriptase from human immunodeficiency virus type 2, M-MLVreverse transcriptase from the Moloney murine leukemia virus, AMVreverse transcriptase from the avian myeloblastosis virus, or Telomerasereverse transcriptase. In embodiments, the depletion polymerase is anucleotide cyclase. In embodiments, the depletion polymerase is aterminal transferase (e.g., terminal deoxycytidyl transferase orterminal deoxynucleotidyl transferase (TdT)). In embodiments, thedepletion polymerase is an RNA dependent polymerase. In embodiments, thedepletion polymerase is a Klenow fragment or mutant thereof, solubleguanylyl cyclase or mutant thereof, or a terminal deoxynucleotidyltransferase (TdT).

In an aspect is provided a method of determining the sequence of atarget single-stranded polynucleotide. In embodiments, the methodincludes incorporating a compound as described herein, (e.g., a compoundof Formula I or Formula II) into an oligonucleotide strand complementaryto at least a portion of the target polynucleotide strand; and detectingthe identity of the compound incorporated into the oligonucleotidestrand. In embodiments, the compound includes a3′-O-polymerase-compatible cleavable moiety as described herein and adetectable label. In embodiments, the method further includes chemicallyremoving the detectable label and the 3′-O-polymerase-compatiblecleavable moiety from the compound incorporated into the oligonucleotidestrand. In embodiments, the 3′-O-polymerase-compatible cleavable moietyand the detectable label of the incorporated compound are removed priorto introducing the next complementary compound. In embodiments, the3′-O-polymerase-compatible cleavable moiety and the detectable label areremoved in a single step of chemical reaction. In embodiments, thesequential incorporation described herein is performed at least 50times, at least 100 times, at least 150 times, at least 200 times, atleast 250 times, at least 300 times, at least 350 times, at least 400times, at least 450 times, or at least 500 times. In embodiments, thesequential incorporation is performed 80 to 200 times. In embodiments,the sequential incorporation is performed 100 to 200 times. Inembodiments, the sequential incorporation is performed 120 to 250 times.In embodiments, the sequential incorporation is performed 100 to 300times. In embodiments, the sequential incorporation is performed 100 to500 times. In embodiments, the sequential incorporation is performed 100to 800 times.

In embodiments, chemical cleavage of a compound (e.g., cleavage of apolymerase-compatible cleavable moiety or cleavage of a linker of acompound described herein) described herein (e.g., in an aspect orembodiment) includes contacting the compound with a reducing agent. Inembodiments, chemical cleavage of a (e.g., cleavage of apolymerase-compatible cleavable moiety or cleavage of a linker of acompound described herein) described herein (e.g., in an aspect orembodiment) includes contacting the compound with THPP (e.g., about 10mM THPP, at least 10 mM THPP). In embodiments, chemical cleavage of acompound (e.g., cleavage of a polymerase-compatible cleavable moiety orcleavage of a linker of a compound described herein) described herein(e.g., in an aspect or embodiment) is performed at about 55 degreesCelsius. In embodiments, chemical cleavage of a compound (e.g., cleavageof a polymerase-compatible cleavable moiety or cleavage of a linker of acompound described herein) described herein (e.g., in an aspect orembodiment) is performed at a temperature of at least 55 degreesCelsius. In embodiments, chemical cleavage of a compound (e.g., cleavageof a polymerase-compatible cleavable moiety or cleavage of a linker of acompound described herein) described herein (e.g., in an aspect orembodiment) is performed at about pH 9.5 to 10.0. In embodiments,chemical cleavage of a compound (e.g., cleavage of apolymerase-compatible cleavable moiety or cleavage of a linker of acompound described herein) described herein (e.g., in an aspect orembodiment) is performed at a pH from 9.5 to 10.0. In embodiments,chemical cleavage of a compound (e.g., cleavage of apolymerase-compatible cleavable moiety or cleavage of a linker of acompound described herein) described herein (e.g., in an aspect orembodiment) is performed at pH 9.5.

In an aspect is a method of incorporating a compound into a primer, themethod including combining a polymerase, a primer hybridized to nucleicacid template and the compound within a reaction vessel and allowing thepolymerase to incorporate the compound into the primer thereby formingan extended primer, wherein the compound is a compound as describedherein and in related embodiments. In embodiments, the method includesdetecting the compound (e.g., detecting the detectable moiety). Inembodiments, the method includes removing the detectable moiety.Sequencing includes, for example, detecting a sequence of signals.Examples of sequencing include, but are not limited to, sequencing bysynthesis (SBS) processes in which reversibly terminated nucleotidescarrying fluorescent dyes are incorporated into a growing strand,complementary to the target strand being sequenced. In embodiments, thenucleotides are labeled with up to four unique fluorescent dyes. Inembodiments, the nucleotides are labeled with at least two uniquefluorescent dyes. In embodiments, the readout is accomplished byepifluorescence imaging. A variety of sequencing chemistries areavailable, non-limiting examples of which are described herein.

In embodiments, the methods (e.g., methods of incorporating a compoundinto a primer and/or methods of sequencing) herein are performed in situon isolated cells or in tissue sections that have been preparedaccording to methodologies known in the art. Methods forpermeabilization and fixation of cells and tissue samples are known inthe art, as exemplified by Cremer et al., The Nucleus: Volume 1: Nucleiand Subnuclear Components, R. Hancock (ed.) 2008; and Larsson et al.,Nat. Methods (2010) 7:395-397, the content of each of which isincorporated herein by reference in its entirety. In embodiments, thecell is cleared (e.g., digested) of proteins, lipids, or proteins andlipids.

In embodiments, the cell in situ is obtained from a subject (e.g., humanor animal tissue). Once obtained, the cell is placed in an artificialenvironment in plastic or glass containers supported with specializedmedium containing essential nutrients and growth factors to supportproliferation. In embodiments, the cell is permeabilized and immobilizedto a solid support surface. In embodiments, the cell is permeabilizedand immobilized to an array (i.e., to discrete locations arranged in anarray). In embodiments, the cell is immobilized to a solid supportsurface. In embodiments, the surface includes a patterned surface (e.g.,suitable for immobilization of a plurality of cells in an orderedpattern. In embodiments, a plurality of cells are immobilized on apatterned surface that have a mean or median separation from one anotherof about 10-20 μm. In embodiments, a plurality of cells are immobilizedon a patterned surface that have a mean or median separation from oneanother of about 1-10 μm. In embodiments, a plurality of cells areimmobilized on a patterned surface that have a mean or median separationfrom one another of about 10-20; 10-50; or 100 μm. In embodiments, aplurality of cells are arrayed on a substrate. In embodiments, aplurality of cells are immobilized in a 96-well microplate having a meanor median well-to-well spacing of about 8 mm to about 12 mm (e.g., about9 mm). In embodiments, a plurality of cells are immobilized in a384-well microplate having a mean or median well-to-well spacing ofabout 3 mm to about 6 mm (e.g., about 4.5 mm).

EXAMPLES Example 1. Novel Modified Nucleotides

DNA sequencing is a fundamental tool in biological and medical research;it is an essential technology for the paradigm of personalized precisionmedicine. Sanger sequencing, where the sequence of a nucleic acid isdetermined by selective incorporation and detection ofdideoxynucleotides, enabled the mapping of the first human referencegenome. While this methodology is still useful for validating newersequencing technologies, efforts to sequence and assemble genomes usingthe Sanger method are an expensive and laborious undertaking, requiringspecialized equipment and expertise. Certain new sequencingmethodologies make use of simultaneously sequencing millions offragments of nucleic acids, resulting in a 50,000-fold drop in the costsassociated with sequencing.

Traditional sequencing-by-synthesis (SBS) methodologies employ serialincorporation and detection of labeled nucleotide analogues. Forexample, high-throughput SBS technology uses cleavable fluorescentnucleotide reversible terminator (NRT) sequencing chemistry. Thesecleavable fluorescent NRTs were designed based on the followingrationale: each of the four nucleotides (A, C, G, T, and/or U) ismodified by attaching a unique cleavable fluorophore to the specificlocation of the nucleobase and capping the 3′—OH group of the nucleotidesugar with a small reversible moiety (also referred to herein as areversible terminator) so that they are still recognized by DNApolymerase as substrates. The reversible terminator (also referred toherein as a polymerase-compatible cleavable moiety) temporarily haltsthe polymerase reaction after nucleotide incorporation while thefluorophore signal is detected. After incorporation and signaldetection, the fluorophore and the reversible terminator are cleaved toresume the polymerase reaction in the next cycle. A balance needs to befound between efficient incorporation of the labeled nucleotides,efficient cleavage to remove all the incorporated labels, and efficientincorporation of the next nucleotide. Described herein in are optimizednucleotide structures and synthetic schemes that improve the performanceof nucleotides in Sequencing-by-Synthesis (SBS) cycles.

Sequencing by synthesis of nucleic acids ideally requires the controlled(i.e., one at a time), yet rapid, incorporation of the correctcomplementary nucleotide opposite the oligonucleotide being sequenced.This allows for accurate sequencing by adding nucleotides in multiplecycles as each nucleotide residue is sequenced one at a time, thuspreventing an uncontrolled series of incorporations occurring.Nucleotides bearing a 3′ RT have been described in the literature, seefor example U.S. Pat. No. 6,664,079 or Ju J. et al. (2006) Proc NatlAcad. Sci USA 103(52):19635-19640, however despite the recent advancesonly a few solutions have been presented, most of which cause otherproblems, including inefficient or incomplete incorporation by thepolymerase, inefficient or incomplete cleavage of the removable group,or harsh conditions needed for the cleaving step causing spuriousproblems with the remainder of the assay and/or fidelity of the targetsequence.

An important feature of a NRT is a detectable moiety, such as afluorophore, that can be cleaved to release the detectable moiety, whichindicates incorporation of the nucleotide. The detectable moiety can beattached to the nucleotide through a cleavable linker. The use of acleavable linker ensures that if required, the label can be removedafter detection. Suitable linkers can be adapted from standard chemicalblocking groups, as disclosed in Greene & Wuts, Protective Groups inOrganic Synthesis, John Wiley & Sons and in Guillier et al (Chem Rev,100: 2092-2157, 2000). For example, a linker used in SBS methodologiesis a linear disulfide linker (referred to herein as an SS linker) havingthe formula:

wherein the SS linker is linear (i.e., continuous) within the linker.Upon exposure to a reducing agent, the nucleotide intermediate includesa reactive thiol capable of interacting with the polymerase or otherreactive moieties and decreasing the efficiency. In contrast, certaincompounds described herein include a Si-trigger linker and/or aSi-trigger RT, which forms non-reactive nucleotides and cleavesapproximately 5× faster than the SS linker or RT under similarconditions. In embodiments, modifying the reaction conditions (e.g.,elevating the temperature to 65° C., increasing the pH) results infaster cleavage.

The compounds described herein include a cleavable site, that uponcontact with a suitable reducing agent, reacts to form a nucleotideterminating with a hydroxyl group rather than a thiol group. Productslinked to the nucleotide having a free hydroxyl group are far lesssusceptible to side reactions with other nucleotides or proteins in situthan products with a free thiol group since a free hydroxyl group isless nucleophilic and acid than a free thiol group (see Principles ofOrganic Chemistry, Ouellette and Rawn, 2015, Elsevier, p. 194-195).

Minimal scar nucleotides. Following detecting and identifying theincorporated nucleotide (e.g., the nucleotide as described herein), thelinker can be cleaved thus allowing the fluorophore to be removed.Cleavage may result in a “scar” moiety located on each of the detectednucleotides, which may negatively impact incorporation of the subsequentnucleotide. Minimizing the scar length can improve sequencing results byallowing for more efficient nucleotide incorporation.

Example 2. Cleavage Kinetics and Thioaldehyde Stability

The speed of sequencing cycle (e.g., the time to incorporate, detect,and cleave a modified nucleotide) is inherently limited by the reactionkinetics. Improvements in sequencing cycle times may be realized if thekinetics of cleaving the linker and/or the reversible terminator of alabeled modified nucleotide is increased. The kinetics of a reactiondepend on the activation energy, i.e., the difference between the energyof the reactants and the transition state. However, transition stateshave only a transitory existence and are difficult, if not impossible,to observe, isolate, and quantify. Reaction rates can generally bepredicted by the Hammond Postulate, which suggests that the activationenergy of the rate-determining step is inversely proportional to thestability of the transition state. The Hammond Postulate stipulates thatthe more geometrically similar the the transition state generated is tothe product, the more quickly the reaction should progress (see forexample March's Advanced Organic Chemistry, 6th Ed., Wiley, 2007,Michael B. Smith and Jerry March, Chapter 6 Methods of determiningmechanisms, page 308). In an endothermic reaction the transition statestructure is closer to the structure of the products, and so it followsthat a more stable product reflects a more stable transition state andhas a lower activation energy.

In the compounds described herein, following the cleavage of thedisulfide bond, the resultant thiol possesses transitory existencebefore a tandem nucleophilic fragmentation reaction yields athioaldehyde. Shown in Scheme 2 is a proposed mechanism for formation ofthe thioaldehyde products generated following cleavage of the disulfidebond.

By invoking the Hammond Postulate, one would expect that any change thathelps to stabilize the products relative to the reactants, which wouldincrease the thermodynamic stability of the resultant thioaldehyde,would also influence the cleavage rate. Simple thermodynamics providesthe enthalpy changes of the reaction, ΔH, as a measure of thethermodynamic stability. The enthalpy change is calculated as thedifference in the enthalpy of the products and reactants,ΔH=ΔH_(products)−ΔH_(reactants). Given the proposed reaction mechanismand suggested transition state (TS), a thioaldehyde with a substituent(R) that better stabilizes the sp² carbon should result in a more stableTS and permit for faster cleavage since the kinetics of a reactiondepend on the activation energy, i.e., the difference between the energyof the reactants and the transition state. The stability of theresultant thioaldehyde influences the cleavage rate of the thioacetal,and was experimentally confirmed in data presented in U.S. Pat. No.11,174,281, which is incorporated herein by reference in its entirety.Briefly, for a nucleotide having a reversible terminator (RT) with thestructure

(referred to as a methylene disulfide, or RT #1) wherein the oxygen isattached to the 3′ position of the deoxyribose, cleavage with a reducingagent results in the formation of thioformaldehyde, a notoriouslyunstable molecule which rapidly oligomerizes to 1,3,5-trithiane.Thioformaldehydes are highly reactive and inherently unstable speciesdue to the lack of steric and resonance stabilization afforded to thesp² carbon by the hydrogens. In accordance with the Hammond Postulate,the transition state is geometrically more similar to the thioaldehydefor this particular reaction (see for example March's Advanced OrganicChemistry, 6th Ed., Wiley, 2007, Michael B. Smith and Jerry March,Chapter 6 Methods of determining mechanisms, page 308). Conceptualizingthe thioaldehyde with all available resonance geometries suggests astable ylide structure that is geometrically similar to the resultantthioaldehyde will be more thermodynamically favored. Therefore,increasing the resonance stabilization to the sp² carbon by including aresonance-stabilizing moiety involves only a small reorganization of themolecular structures and thus permits faster cleavage. For example, anRT that includes a methyl substituent on the methylene carbon having thestructure

(RT #2) increases the cleavage rate approximately 10-fold relative to RT#1. Modulating the resonance stabilization (e.g., replacing the methylwith a cyclic moiety, such as a phenyl) further increased the cleavagekinetics approximately 13-fold. Thus, a substituent that betterstabilizes the sp² carbon results in a transitional state that is morethermodynamically favored and geometrically similar to the resultantthioaldehyde, enabling a faster reaction. Stabilization of carbocationsthrough donation of π electrons from adjacent carbon orbitals fromspecies such as allyl and benzyl cations is known. Another form ofstabilization of carbocations can occur through filled σ orbitals, whichcan donate electrons to an empty π orbital, referred to ashyperconjugation.

Silicon hyperconjugation is a unique type of hyperconjugation thatdescribes the stabilizing influence of a silicon atom on the developmentof positive charge at a carbon atom one position removed, referred to asthe (3 position, from the silicon atom, (see Wierschke et al. JACS 1985,107, 1496-4500 and Lambert et al, Acc. Chem. Res., 1999, 32(2), p.183-190). The β-silicon effect refers to the ability of silyl groupsthat are positioned (3 to the carbocation (C⁺) to promote the formationor development of carbenium ions due to the overlapping C—Si bondorbital and empty p_(π) orbital of the carbenium ion, either by bridgingor by hyperconjugation. Thus, the electrons found in the C—Si bondorbital overlap and can delocalize into the empty p_(π) orbital of thecarbenium, helping to stabilize the transition state. Theelectropositive nature of silicon helps to contribute to this effect.Therefore, the activation energy of the reaction is lower, resulting infaster cleavage and generation of the product.

A model compound representing the Si-trigger linkers and Si-triggerreversible terminator having the general formula:

wherein the R corresponds to the substituent provided in Table 2, wasused for computational analysis. Theoretical calculations of the changein enthalpy, ΔH=ΔH_(products)−ΔH_(reactants), for the fragmentationreaction depicted in Scheme 2 was performed to predict which disulfidebonds will cleave rapidly under suitable conditions. Gas phasecalculations were performed using hybrid Density Functional Theory(B3LYP) with a large basis set (Valence triple-zeta with two sets ofpolarization functions). The optimized structure and energy of thereactants and the products were determined and calculated ΔH for avariety of compounds is shown in Table 2.

TABLE 2 Calculated enthalpies for reaction described in Scheme 2. Thecleavage rate was determined by contacting the compounds with 0.1-1.0 mMTHPP at 55° C. dH Cleavage dH Cleavage R (kcal/mol) rate(s) R (kcal/mol)rate(s) H 18.0   95 +/− 3 6.6 CN 12.4 

9.2 phenyl 5.6  1.8 +/− 3

10.4  CH₃ 13.5  12.6 +/− 3

14.7 

13.4 

6.4

8.2

5.4

Using ΔH as a corollary for the reaction rate, it is possible to predictwhich disulfide linkers will cleave more rapidly under suitableconditions. We found that in general, reducing the energetic burden onthe system, i.e., reducing the enthalpy, correlates with fastercleavage. Experimental evidence supports using ΔH as a proxy for thereaction rate, as reported in Table 2 and, showing the experimentallyderived cleavage halftime when R is H, methyl, and phenyl decreases asthe calculated ΔH decreases. Reducing the energetic burden on thesystem, i.e., reducing the enthalpy, corresponds to faster cleavagerates.

The calculated values of ΔH suggest that a linker having an Si atompositioned β (dH 8.2 kcal/mol) rather than a (dH 13.4 kcal/mol) to thegenerated carbocation, cleaves faster. The decreased enthalpy ΔH is dueto the aforementioned β-silicon effect, which predicts that Sistabilizes the transition state. Further since the ΔH of R═CH₂SiMe₃ (8.2kcal/mol) falls between the calculated value of ΔH when R=phenyl (5.6kcal/mol) and R=methyl (13.5 kcal/mol), this suggests that compoundswith a disulfide linkage having R═CH₂SiMe₃ cleave at a rate faster thanwhen R is CH₃. Therefore, the modified compounds as described herein arestable and have Si containing disulfide linkers that can rapidly cleavedunder mild conditions.

Example 3. Chemical Synthesis of Modified Nucleotides

Described herein is a generalized process for synthesizing compounds offormula

wherein R³ is a —O-polymerase compatible cleavable moiety having theformula:

Compounds synthesized in Scheme 4 are further modified as shown inScheme 5 to incorporate the disulfide group to provide compounds with R³as a —O-polymerase compatible cleavable moiety having the formula:

wherein R², R⁴, R⁵, R₆, R₇, R₈, R₉, R¹⁰, L¹⁰⁰ and B are as describedherein.

The compounds in Scheme 5 having the —S—S— with methyl substituent maybe modified according to Scheme 6.

What is claimed is:
 1. A compound having the formula:

wherein B is a divalent nucleobase; R¹ is a 5′-nucleoside protectinggroup, monophosphate moiety, polyphosphate moiety, or nucleic acidmoiety; R¹ and R³ are independently hydrogen, halogen, —CCl₃, —CBr₃,—CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,polymerase-compatible cleavable moiety, or an —O-polymerase-compatiblecleavable moiety; R⁴ is a detectable moiety; and L¹⁰⁰ is a divalentlinker comprising

wherein R⁵ and R⁶ are independently hydrogen, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; and R⁷, R⁸, and R⁹ are independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.
 2. The compound of claim 1,wherein R¹ is hydrogen.
 3. The compound of claim 1, wherein R¹ is atriphosphate moiety.
 4. The compound of claim 1, wherein B is a cytosineor a derivative thereof, guanine or a derivative thereof, adenine or aderivative thereof, thymine or a derivative thereof, uracil or aderivative thereof, hypoxanthine or a derivative thereof, xanthine or aderivative thereof, 7-methylguanine or a derivative thereof,5,6-dihydrouracil or a derivative thereof, 5-methylcytosine or aderivative thereof, or 5-hydroxymethylcytosine or a derivative thereof.5. The compound of claim 1, having the formula:


6. The compound of claim 1, wherein L¹⁰⁰ is -L¹⁰¹-L¹⁰²-L¹⁰³-L¹⁰⁴-L¹⁰⁵-;L¹⁰¹, L¹⁰², L¹⁰⁴, and L¹⁰⁵ are independently a bond, —NH—, —O—, —C(O)—,—C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; and L¹⁰³ is


7. The compound of claim 1, wherein R⁵ and R⁶ are independentlyhydrogen, or substituted or unsubstituted alkyl.
 8. The compound ofclaim 1, wherein R⁵ and R⁶ are independently hydrogen, or unsubstitutedC₁-C₄ alkyl.
 9. The compound of claim 1, wherein R⁷, R⁸, and R⁹ areindependently substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted 2 to 8 membered heteroalkyl, substituted or unsubstitutedC₃-C₆ cycloalkyl, substituted or unsubstituted 2 to 8 memberedheterocycloalkyl, substituted or unsubstituted C₆ to C₁₂ aryl, orsubstituted or unsubstituted 2 to 8 membered heteroaryl.
 10. Thecompound of claim 1, wherein R⁷, R⁸, and R⁹ are independentlysubstituted or unsubstituted C₁-C₆ alkyl.
 11. The compound of claim 1,wherein R⁷, R⁸, and R⁹ are unsubstituted C₁-C₆ alkyl.
 12. The compoundof claim 6, wherein L¹⁰⁰ has the formula:


13. The compound of claim 6, wherein L¹⁰⁰ has the formula:


14. The compound of claim 1, wherein R³ is an —O-polymerase-compatiblecleavable moiety having the formula

wherein R⁶ is substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl; and R¹⁰ issubstituted or unsubstituted alkyl.
 15. A compound having the formula:

wherein B is a divalent nucleobase; R¹ is a 5′-nucleoside protectinggroup, monophosphate moiety, polyphosphate moiety, or nucleic acidmoiety; R² is hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCl₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl, or apolymerase-compatible cleavable moiety; R¹ is

R⁴ is a detectable moiety; L¹⁰⁰ is a divalent linker; R⁵ and R⁶ areindependently hydrogen, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; R⁷,R⁸, and R⁹ are independently hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl; and R¹⁰ is substituted or unsubstituted alkyl.
 16. A methodfor sequencing a nucleic acid, comprising: incorporating in series witha nucleic acid polymerase, within a reaction vessel, one of fourdifferent compounds into a primer to create an extension strand, whereinsaid primer is hybridized to said nucleic acid and wherein each of thefour different compounds comprises a unique detectable label; (ii)detecting said unique detectable label of each incorporated compound, soas to thereby identify each incorporated compound in said extensionstrand, thereby sequencing the nucleic acid; wherein each of said fourdifferent compounds is independently a compound of claim
 1. 17. A methodfor sequencing a nucleic acid, comprising: (i) incorporating in serieswith a nucleic acid polymerase, within a reaction vessel, one of fourdifferent compounds into a primer to create an extension strand, whereinsaid primer is hybridized to said nucleic acid and wherein each of thefour different compounds comprises a unique detectable label; (ii)detecting said unique detectable label of each incorporated compound, soas to thereby identify each incorporated compound in said extensionstrand, thereby sequencing the nucleic acid; wherein each of said fourdifferent compounds is independently a compound of claim
 15. 18. Amethod of sequencing nucleic acid comprising: (i) providing a nucleicacid template hybridized to a primer; (ii) extending the primerhybridized to said nucleic acid template with a compound of claim 1; and(iii) identifying the compound, so as to sequence the nucleic acid. 19.A method of incorporating a compound into a primer, the methodcomprising combining a polymerase, a primer hybridized to nucleic acidtemplate and the compound within a reaction vessel and allowing saidpolymerase to incorporate said compound into said primer thereby formingan extended primer, wherein said compound is a compound of claim
 1. 20.A nucleic acid polymerase complex comprising a nucleic acid polymerase,wherein said nucleic acid polymerase is bound to a compound of claim 1.